U.S. patent application number 16/445130 was filed with the patent office on 2019-10-10 for methods and compositions for the treatment of persistent infections and cancer by inhibiting the programmed cell death 1 (pd-1) .
This patent application is currently assigned to Emory University. The applicant listed for this patent is Brigham and Women's Hospital, Inc., Dana-Farber Cancer Institute, Inc., Emory University, President and Fellows of Harvard College. Invention is credited to Rafi Ahmed, Daniel Barber, David M. Dorfman, Gordon Freeman, Arlene Sharpe, E. John Wherry.
Application Number | 20190309065 16/445130 |
Document ID | / |
Family ID | 37499143 |
Filed Date | 2019-10-10 |
United States Patent
Application |
20190309065 |
Kind Code |
A1 |
Freeman; Gordon ; et
al. |
October 10, 2019 |
METHODS AND COMPOSITIONS FOR THE TREATMENT OF PERSISTENT INFECTIONS
AND CANCER BY INHIBITING THE PROGRAMMED CELL DEATH 1 (PD-1)
PATHWAY
Abstract
The present invention provides methods and compositions for the
treatment, prevention, or reduction of persistent infections, such
as chronic infections, latent infections, and slow infections and
cancer. The methods and compositions of the invention are also
useful for the alleviation of one or more symptoms associated with
such infections and cancer.
Inventors: |
Freeman; Gordon; (Brookline,
MA) ; Sharpe; Arlene; (Brookline, MA) ;
Dorfman; David M.; (Brookline, MA) ; Ahmed; Rafi;
(Atlanta, GA) ; Barber; Daniel; (Rockville,
MD) ; Wherry; E. John; (Havertown, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Emory University
Dana-Farber Cancer Institute, Inc.
Brigham and Women's Hospital, Inc.
President and Fellows of Harvard College |
Atlanta
Boston
Boston
Cambridge |
GA
MA
MA
MA |
US
US
US
US |
|
|
Assignee: |
Emory University
Atlanta
GA
Dana-Farber Cancer Institute, Inc.
Boston
MA
Brigham and Women's Hospital, Inc.
Boston
MA
President and Fellows of Harvard College
Cambridge
MA
|
Family ID: |
37499143 |
Appl. No.: |
16/445130 |
Filed: |
June 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15252133 |
Aug 30, 2016 |
10370446 |
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16445130 |
|
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|
|
14144304 |
Dec 30, 2013 |
9457080 |
|
|
15252133 |
|
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11449919 |
Jun 8, 2006 |
8652465 |
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14144304 |
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60688872 |
Jun 8, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/39 20130101;
A61P 33/06 20180101; A61P 35/00 20180101; C07K 2317/74 20130101;
G01N 2800/26 20130101; Y02A 50/30 20180101; G01N 33/57484 20130101;
C07K 16/2818 20130101; A61P 31/00 20180101; Y02A 50/467 20180101;
A61K 2039/507 20130101; A61P 43/00 20180101; A61P 37/04 20180101;
A61P 31/22 20180101; C12N 15/1138 20130101; A61K 39/39558 20130101;
A61K 45/06 20130101; A61K 2039/572 20130101; C07K 16/2827 20130101;
A61K 39/3955 20130101; A61K 2039/55516 20130101; C07K 2317/76
20130101; A61P 31/14 20180101; G01N 33/57492 20130101; G01N 33/505
20130101; A61P 1/16 20180101; A61P 33/00 20180101; C07K 2317/73
20130101; C12N 2310/14 20130101; A61K 31/713 20130101; A61K
2039/505 20130101; A61K 2039/55566 20130101; A61P 31/06 20180101;
A61P 31/10 20180101; Y02A 50/58 20180101; A61P 31/20 20180101; A61K
38/1709 20130101; C07K 2317/70 20130101; A61P 31/04 20180101; A61P
31/12 20180101; G01N 33/5011 20130101; C07K 16/2803 20130101; A61P
31/18 20180101; Y02A 50/412 20180101; A61P 33/02 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/574 20060101 G01N033/574; A61K 38/17 20060101
A61K038/17; G01N 33/50 20060101 G01N033/50; A61K 39/39 20060101
A61K039/39; A61K 31/713 20060101 A61K031/713; A61K 45/06 20060101
A61K045/06 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under
AI039671 and CA084500 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A method of increasing cytotoxic activity of anergic T cells in
a subject having a lymphoproliferative cancer and/or decreasing a
symptom of the lymphoproliferative cancer in the subject,
comprising administering to the subject having the
lymphoproliferative cancer an effective amount of an agent that
reduces the activity of a Programmed Cell Death (PD)-1 polypeptide,
wherein the agent is an anti-PD-1 antibody, thereby increasing the
cytotoxic activity of anergic T cells and/or decreasing a symptom
of the lymphoproliferative cancer in the subject, wherein the
lymphoproliferative cancer is an angioimmunoblastic lymphoma.
2. The method of claim 1, wherein increasing the cytotoxic activity
of anergic T cells comprises increasing cytokine production or T
cell proliferation.
3. The method of claim 2, wherein the cytokine is interferon
.gamma., tumor necrosis factor .alpha. or interleukin-2.
4. The method of claim 1, further comprising measuring cytotoxic T
cell activity in a biological sample from the subject.
5. The method of claim 4, wherein measuring cytotoxic T cell
activity comprises: a) measuring the cytotoxic activity of anergic
CD8+ T cells in the biological sample from the subject; or b)
measuring the production of a cytokine by T cells in the biological
sample from the subject.
6. The method of claim 5, wherein the cytokine is interferon
.gamma., tumor necrosis factor .alpha. or interleukin-2.
7. The method of claim 1, wherein the method further comprises
administering to the subject an effective amount of a second
compound that induces an immune response.
8. The compound of claim 7, wherein the second compound is an
anti-inflammatory compound, an antineoplastic compound or an
analgesic.
9. The method of claim 7, wherein the second compound is an
anti-CTLA-4 antibody, an anti-BTLA antibody, or an anti-B7-H4
antibody.
10. The method of claim 7, wherein the second compound reduces
cancer cell volume.
11. The method of claim 1, wherein the antibody is a monoclonal
antibody, a humanized antibody, a deimmunized antibody, or an
immunoglobulin (Ig) fusion protein.
12. The method of claim 1, wherein the subject is human.
13. The method of claim 1, wherein the method decreases the symptom
of the lymphoproliferative cancer in the subject.
14. The method of claim 1, wherein the method decreases the size of
the tumor or the prevalence of cancer cells.
15. A method of increasing cytotoxic activity of anergic T cells in
a subject having a lymphoproliferative cancer, and/or decreasing
the symptom of the lymphoproliferative cancer in the subject,
comprising administering to the subject having the
lymphoproliferative cancer an effective amount of (a) an agent that
reduces the activity of a Programmed Cell Death (PD)-1 polypeptide,
wherein the agent is an anti-PD-1 antibody or an anti-Programmed
Cell Death Ligand (PD-L1) antibody, and (b) an anti-CTLA-4
antibody, thereby increasing the cytotoxic activity of anergic T
cells in the subject with the lymphoproliferative cancer and/or
decreasing the symptom of the lymphoproliferative cancer, wherein
the lymphoproliferative cancer is a Hodgkin's lymphoma or
angioimmunoblastic T cell lymphoma.
16. The method of claim 15, wherein the lymphoproliferative cancer
is the Hodgkin's lymphoma.
17. The method of claim 15, wherein the Hodgkin's lymphoma is the
is a nodular lymphocyte predominant Hodgkin's lymphoma.
18. The method of claim 15, wherein the subject is human.
19. The method of claim 15, wherein the lymphoproliferative cancer
is the angioimmunoblastic lymphoma, and wherein the method reduces
the symptom of the angioimmunoblastic lymphoma.
20. The method of claim 15, wherein a) the anti-PD-L1 antibody is a
monoclonal antibody, a humanized antibody, a deimmunized antibody,
or an immunoglobulin (Ig) fusion protein; or b) the anti-PD-1
antibody is a monoclonal antibody, a humanized antibody, a
deimmunized antibody, or an Ig fusion protein.
Description
RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent Ser. No. 15/252,133,
filed on Aug. 30, 2016, which is a continuation of U.S. patent
application Ser. No. 14/144,304, filed on Dec. 30, 2013, issued as
U.S. Pat. No. 9,457,080, which is a continuation of U.S. patent
application Ser. No. 11/449,919, filed on Jun. 8, 2006, issued as
U.S. Pat. No. 8,652,465, which claims the benefit of U.S.
Provisional Application No. 60/688,872, filed on Jun. 8, 2005. The
prior applications are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0003] In general, the present invention relates to methods and
compositions for the treatment of persistent infections and
cancer.
BACKGROUND OF THE INVENTION
[0004] Although the development of preventative vaccines has
significantly reduced the mortality rate of viral infections, the
use of such vaccines against viruses that cause persistent
infections (e.g., hepatitis C virus) has been met with limited
success. In contrast to viruses that cause acute and self-limited
infections, the immune response that is mounted against persistent
infection-causing microbes is often transient and insufficient to
clear the infection. As a result, the infectious microbe remains
within the infected subject for extended periods of time, without
necessarily causing constant host damage.
[0005] A major impediment in the eradication of persistent
infection-causing microbes is the ability of such microbes to evade
the immune system of the host organism. For example, certain
viruses and parasites down-regulate the expression of host
molecules necessary for efficient T cell recognition of infected
cells. Persistent infections also cause the functional impairment
of antigen specific CD8+ T cells, which are vital to the control
and eradication of viral infections. Although the combination of
therapeutic vaccines with cytokine adjuvants has been encouraging,
the resulting immune responses have not successfully eradicated the
pathogen.
[0006] Thus, better methods are needed to treat, prevent, or
alleviate persistent infections.
SUMMARY OF THE INVENTION
[0007] The present invention provides methods and compositions for
the treatment, prevention, or reduction of, or alternatively the
alleviation of one or more symptoms of, a persistent infection or
cancer. The invention is based on the discovery that antigen
specific CD8+ T cells become functionally tolerant (`exhausted`) to
the infectious agent following the induction of the Programmed
Death-1 polypeptide (PD-1). Accordingly, by reducing the expression
or activity of PD-1, PD-L1 or PDL2, the proliferation of
functionally tolerant CD8+ T cells, the production of cytokines,
and the rate of an infectious agent (e.g., viral, bacterial,
fungal, parasite, mycoplasm or cancer) clearance is increased such
that the immune response specific to the infectious agent is
enhanced.
[0008] Accordingly, the invention provides a method of alleviating
or preventing a symptom of a persistent infection (e.g., a viral
infection, a bacterial infection, a fungal infection, a mycoplasm
infection and a parasitic infection) or cancer by administering to
a subject in need thereof (e.g., a human) a compound that reduces
the activity or expression of a member of the CD28-like family
(e.g., PD-1, CTLA-4, BTLA and a functional fragment or variant
thereof) or CD28-like family ligands (e.g., PD-L1 or PD-L2).
Alternatively, the subject is administered an antigen-specific T
cell or B cell that has been contacted with an compound that
reduces the expression or activity of a PD-1 polypeptide in the
cell. For example, the antigen specific T cell or B cell is
specific to a viral antigen. The T cell or B cell is derived from
an autologous source or is derived from another subject of the same
or different species as the subject being treated.
[0009] In addition, the invention features a method of increasing
the cytotoxic activity of a T cell (e.g., anergic T cell or T cell
having increased tolerance to antigens) by contacting the T cell
with a compound that reduces the activity or expression of a PD-1
polypeptide.
[0010] In all foregoing aspects of the invention, persistent viral
infections result from infections such as a hepatitis virus, a
human immunodeficiency virus (HIV), a human T-lymphotrophic virus
(HTLV), a herpes virus, an Epstein-Barr virus, or a human papilloma
virus. Persistent viral infections may also include infections
caused by a latent virus. Cancers include lymphoproliferative
disorders such as angioimmunoblastic lymphoma and nodular
lymphocyte Hodgkin lymphoma. Desirably, the compound of the
invention increases an antigen specific immune response by
increasing the cytotoxic T-cell activity (e.g., an increase in
cytotoxic cytokine production such as IFN.gamma., TNF.alpha., or
IL-2, an increase in T cell proliferation, or an increase in viral
clearance) in the subject being treated. For example, the compound
reduces the expression or activity of a PD ligand 1 (PD-L1) or a PD
ligand 2 (PD-L2) or reduces the interaction between PD-1 and PD-L1
or the interaction between PD-1 and PD-L2. Exemplary compounds
include antibodies (e.g., an anti-PD-1 antibody, an anti-PD-L1
antibody, and an anti-PD-L2 antibody), RNAi molecules (e.g.,
anti-PD-1 RNAi molecules, anti-PD-L1 RNAi, and an anti-PD-L2 RNAi),
antisense molecules (e.g., an anti-PD-1 antisense RNA, an
anti-PD-L1 antisense RNA, and an anti-PD-L2 antisense RNA),
dominant negative proteins (e.g., a dominant negative PD-1 protein,
a dominant negative PD-L1 protein, and a dominant negative PD-L2
protein), and small molecule inhibitors. Antibodies include
monoclonal antibodies, humanized antibodies, deimmunized
antibodies, and Ig fusion proteins. An exemplary anti-PD-L1
antibody includes clone EH12.
[0011] In addition to the compound that reduces PD-1 expression or
activity, the subject being treated may also be administered a
vaccine that may or may not include an adjuvant or a prime booster
shot. Optionally, the subject is administered a second compound,
such as an antiviral compound (e.g., vidarabine, acyclovir,
gancyclovir, valgancyclovir, nucleoside-analog reverse
transcriptase inhibitor (NRTI) such as AZT (Zidovudine), ddI
(Didanosine), ddC (Zalcitabine), d4T (Stavudine), or 3TC
(Lamivudine), non-nucleoside reverse transcriptase inhibitor
(NNRTI) such as nevirapine or delavirdine, protease inhibitor such
as saquinavir, ritonavir, indinavir, or nelfinavir, ribavirin, and
interferon), an antibacterial compound, an antifungal compound, an
antiparasitic compound, an anti-inflammatory compound,
anti-neoplastic compounds or an analgesic. The second compound may
also be a compound that reduces the expression or activity of
cytotoxic T lymphocyte antigen 4 (CTLA-4) or B and T lymphocyte
attenuator (BTLA). Other exemplary compounds that may be
administered to the subject are anti-CTLA-4 antibodies, anti-BTLA
antibodies, anti-CD28 antibodies, anti-ICOS antibodies, anti-ICOS-L
antibodies, anti-B7-1 antibodies, anti-B7-2 antibodies, anti-B7-H3
antibodies, and anti-B7-H4 antibodies.
[0012] The present invention further provides a method for
identifying a candidate compound that modulates the activity or
expression of a PD-1 polypeptide that includes the steps of: (a)
contacting a cell expressing a PD-1 gene (e.g., PD-1 fusion gene)
with a candidate compound; (b) measuring the expression or activity
of PD-1 in the cell (e.g., by measuring the expression of PD-1 mRNA
or protein); and (c) comparing the expression or activity of PD-1
in the cell compared to such expression or activity in a control
cell not contacted with the compound. An increase or decrease in
the expression or activity of PD-1 indicates the candidate compound
as being useful for modulating the activity or expression of a PD-1
polypeptide.
[0013] Alternatively, the screening method may involve the steps
of: (a) contacting a T cell that overexpresses a PD-1 gene with a
candidate compound; and (b) determining the cytotoxic activity of
the T cell; (c) comparing the cytotoxic activity of the T cell
relative to such activity in a control cell not contacted with the
compound. An increase or decrease in such activity identifies the
candidate compound as being useful for modulating the activity or
expression of a PD-1 polypeptide. Cytotoxic activity includes
cytokine production, T cell proliferation, and viral clearance.
[0014] The invention further provides a screening method involving
the steps of: (a) contacting a PD-1 polypeptide with a candidate
compound; (b) determining whether the candidate compound interacts
with the PD-1 polypeptide; and (c) identifying a candidate compound
as useful for modulating PD-1 expression or activity. Desirably,
the identified candidate compound interacts with the PD-1
polypeptide and reduces its activity.
[0015] The candidate compound identified by the screening methods
described herein may reduce the interaction between PD-1 and PD-L1
or the interaction between PD-1 and PD-L2. The cell employed in any
of the screening methods described herein include mammalian cells
such as rodent cells or human cell. The cell is an immune cell,
such as a T cell. Desirably, the PD-1 polypeptide used in such
screening methods is a human PD-1 polypeptide.
[0016] Also provided herein is a method of diagnosing a subject as
having or at risk of having a persistent infection or cancer
involving the steps of: (a) providing a sample containing immune
cells (e.g., T cell or B cell) from a subject, and (b) measuring
the expression or activity of PD-1 in the sample. An increase in
the expression or activity of PD-1 compared to such expression or
activity in a control sample identifies the subject as having or at
risk of having a persistent infection or cancer. Desirably, step
(b) involves identifying antigen-specific immune cells, such as a
viral antigen, bacterial antigen, parasitic antigen, or fungal
antigen.
[0017] A method of selecting a treatment for a subject having or at
risk of having a persistent infection or cancer is also described.
This method involves the steps of: (a) providing a sample
containing immune cells (e.g., T cell or B cell) from a subject;
and (b) measuring the expression or activity of PD-1 in the immune
cells, such that an increase in expression or activity of PD-1
compared to such expression or activity in a control sample
identifies the subject as having or at risk of having a persistent
infection or cancer; and (c) selecting a treatment for the subject
diagnosed as having or at risk a persistent infection or cancer,
such that the treatment includes a compound that reduces the
expression or activity of PD-1. Desirably, step (b) involves
identifying antigen-specific immune cells, such as a viral antigen,
bacterial antigen, parasitic antigen, or fungal antigen.
[0018] Samples derived from subjects include blood samples, tissue
biopsies, and bone marrow samples. Furthermore, control cells may
be derived from a subject that does not have or at risk of having a
persistent infection.
[0019] The invention further provides a composition that contains:
(a) a compound that reduces the level or activity of PD-1; and (b)
a second compound, such as an antiviral compound, an antibacterial
compound, an antifungal compound, an antiparasitic compound, an
anti-inflammatory compound, an analgesic, an anti-CTLA-4 antibody,
an anti-BTLA antibody, an anti-CD28 antibody, an anti-ICOS
antibody, an anti-ICOS-L antibody, an anti-B7-1 antibody, an
anti-B7-2 antibody, an anti-B7-H3 antibody, or an anti-B7-H4
antibody.
[0020] The invention also provides a kit that contains (a) a
compound that reduces the level or activity of PD-1; and (b)
instructions for delivery of the compound to a subject.
Alternatively, the kit contains (a) a first compound that reduces
the level or activity of PD-1; (b) a second compound such as an
antiviral compound, an antibacterial compound, an antifungal
compound, an antiparasitic compound, an anti-inflammatory compound,
an analgesic, an anti-CTLA-4 antibody, an anti-BTLA antibody, an
anti-CD28 antibody, an anti-ICOS antibody, an anti-ICOS-L antibody,
an anti-B7-1 antibody, an anti-B7-2 antibody, an anti-B7-H3
antibody, or an anti-B7-H4 antibody; and (c) instructions for
delivery of the first compound and the second compound to a
subject.
[0021] The present invention provides significant advantages over
standard therapies for treatment, prevention, and reduction, or
alternatively, the alleviation of one or more symptoms of
persistent infections. Administration of the therapeutic agent that
reduces the level or activity of PD-1 increases CD8+ T cell
cytotoxicity, in turn increasing the immune response to the
infectious agent having the ability to establish a persistent
infection. In addition, the candidate compound screening methods
provided by this invention allow for the identification of novel
therapeutics that modify the injury process, rather than merely
mitigating the symptoms.
[0022] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0023] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A is a bar graph showing the levels of PD-1 mRNA in
D.sup.bGP33-41 and/or D.sup.bGP276-286 specific T cells from naive
transgenic mice, lymphocytic choriomeningitis virus (LCMV)
Armstrong immune (approximately 30 days post-infection) infected
mice, or CD4-depleted LCMV-Cl-13 infected mice (approximately 30
days post-infection), as measured by gene array analysis.
[0025] FIG. 1B is a series of images of a flow cytometry experiment
showing PD-1 surface expression on CD8+ tetramer+ T cells in LCMV
Armstrong immune and CD4 depleted LCMV-C1-13 infected mice
approximately 60 days post-infection. Anergic CD8+ T cells express
high levels of PD-1 polypeptide on the cell surface approximately
60 days after chronic infection with LCMV-Cl-13 virus (labeled
"chronic"), but virus-specific CD8+ T cells do not express PD-1
polypeptide after clearance of an acute LCMV Armstrong infection
(labeled "immune").
[0026] FIG. 1C is a series of images of a flow cytometry experiment
demonstrating the presence of PD-L1 on splenocytes from chronically
infected and uninfected mice. It demonstrates that PD-L1 expression
is the highest on the splenocytes that are infected by the
virus.
[0027] FIG. 2A is a series of scatter plots showing that when C1-13
infected mice are treated from day 23 to 37 post-infection there
was approximately a 3 fold increase in the number of DbNP396-404
specific and DbGP33-41 specific CD8 T cells compared to the
untreated controls. In order to determine any changes in function
IFN-.gamma. and TNF-.alpha. production was measured in response to
8 different LCMV epitopes.
[0028] FIG. 2B is a scatter plot showing that when all the known
CD8 T cell specificities are measured there is a 2.3 fold increase
in total number of LCMV specific CD8 T cells.
[0029] FIG. 2C is a series of flow cytometry graphs showing
IFN-.gamma. and TNF-.alpha. production in response to eight
different LCMV epitopes.
[0030] FIG. 2D is a scatter plot showing that more virus specific
CD8 T cells in treated mice have the ability to produce
TNF-.alpha..
[0031] FIG. 2E is a series of bar charts showing that PD-L1
blockade also resulted in increased viral control in the spleen
liver lung and serum.
[0032] FIG. 3A is a graph demonstrating the increase in DbGP33-41
and DbGP276-286 specific CD8+ T cells (labeled "GP33" and "GP276")
in CD4-depleted C1-13 infected mice treated with anti-PD-L1
(labeled ".alpha.PD-L") from day 46 to day 60 post-infection versus
control (labeled "untx"), which demonstrates that mice treated with
anti-PD-L1 contained approximately 7 fold more DbGP276-286 specific
splenic CD8+ T cells and approximately 4 fold more DbGP33-41
specific splenic CD8+ T cells than untreated mice.
[0033] FIG. 3B is a series of images demonstrating the increased
frequency of DbGP33-41 and DbGP276-286 specific CD8+ T cells in the
spleen of CD4-depleted C1-13 infected mice treated with anti-PD-L1
(labeled ".alpha.PD-L1 Tx") from day 46 to day 60 post-infection
versus control (labeled "untx").
[0034] FIG. 3C is a series of images demonstrating increased
proliferation of DbGP276-286 specific CD8+ T cells in
anti-PD-L-treated mice, as measured by BrdU incorporation and Ki67
expression.
[0035] FIG. 3D is a chart showing that mice having high levels of
CD8+ T cell expansion demonstrate an appreciable response in
peripheral blood mononuclear cells (PBMC), as shown by comparing
DbGP276-286 specific CD8+ T cells in the PBMC as compared to
DbGP276-286 specific CD8+ T cells in the spleen.
[0036] FIG. 4A is a series of charts demonstrating the increase in
IFN-.gamma. producing DbGP276-286 and DbGP33-41 specific CD8+ T
cells in anti-PD-L-treated mice, as compared to controls. Higher
frequencies of DbNP396-404, KbNP205-212, DbNP166-175, and
DbGP92-101 specific CD8+ T cells were also detected in
anti-PD-L-treated mice.
[0037] FIG. 4B is a chart demonstrating that in anti-PD-L-treated
mice, 50% of DbGP276-286 specific CD8+ T cells produce IFN-.gamma.,
as compared to 20% of DbGP276-286 specific CD8+ T cells in control
mice.
[0038] FIG. 4C is a series of images demonstrating that
anti-PD-L-treated chronically infected mice produce higher levels
of TNF-.alpha. than untreated chronically infected mice, but still
produce lower levels of TNF-.alpha. than immune mice infected with
LCMV Armstrong virus.
[0039] FIG. 4D is a chart demonstrating that treatment of
LCMV-Cl-13 infected mice with anti-PD-L1 renews ex vivo lytic
activity of the virus-specific T cells, as compared to untreated
infected mice, measured using a .sup.51Cr release assay.
[0040] FIG. 4E is a series of charts demonstrating the reduction of
viral titers in various organs following treatment of LCMV-Cl-13
infected mice with .alpha.-PD-L1. Viral titers decreased
approximately 3 fold in the spleen, 4 fold in the liver, 2 fold in
the lung, and 2 fold in serum after 2 weeks of anti-PD-L1
treatment, as compared to untreated mice.
[0041] FIG. 5A is a series of images of a flow cytometry experiment
showing PD-1 surface expression using 10 HIV tetramers specific for
dominant epitopes targeted in chronic clade C HIV infection. The
percentages indicate the percentage of tetramer.sup.+ cells that
are PD-1.sup.+.
[0042] FIG. 5B is a series of charts demonstrating that the
percentage and MFI of PD-1 is significantly upregulated on
HIV-specific CD8 T cells compared to the total CD8 T cell
population (p<0.0001) in antiretroviral therapy naive
individuals, and PD-1 is increased on the total CD8 T cell
population in HIV-infected versus HIV-seronegative controls
(p=0.0033 and p<0.0001, respectively). 120 HIV tetramer stains
from 65 HIV-infected individuals and 11 HIV seronegative controls
were included in the analysis.
[0043] FIG. 5C is a series of charts showing the median percentage
and MFI of PD-1 expression on tetramer.sup.+ cells by epitope
specificity.
[0044] FIG. 5D is a chart depicting the variation in the percentage
of PD-1.sup.+ cells on different epitope-specific populations
within individuals with multiple detectable responses. Horizontal
bars indicate the median percentage of PD-1.sup.+ HIV
tetramer.sup.+ cells in each individual.
[0045] FIG. 6A is a series of charts demonstrating that there is no
correlation between the number of HIV-specific CD8 T cells, as
measured by tetramer staining, and plasma viral load, whereas there
is a positive correlation between both the percentage and MFI of
PD-1 on tetramer.sup.+ cells and plasma viral load (p=0.0013 and
p<0.0001, respectively).
[0046] FIG. 6B is a series of charts showing that there is no
correlation between the number of HIV tetramer.sup.+ cells and CD4
count, whereas there is an inverse correlation between the
percentage and MFI of PD-1 on HIV tetramer.sup.+ cells and CD4
count (p=0.0046 and p=0.0150, respectively).
[0047] FIG. 6C is a series of charts demonstrating that the
percentage and MFI of PD-1 on the total CD8 T cell population
positively correlate with plasma viral load (p=0.0021 and
p<0.0001, respectively).
[0048] FIG. 6D is a series of charts depicting the percentage and
MFI of PD-1 expression on the total CD8 T cell population is
inversely correlated with CD4 count (p=0.0049 and p=0.0006,
respectively).
[0049] FIG. 7A is a series of images of a flow cytometry experiment
showing representative phenotypic staining of B*4201 TL9-specific
CD8 T cells from subject SK222 in whom 98% of B*4201 TL9-specific
CD8 T cells are PD-1.sup.+.
[0050] FIG. 7B is a chart illustrating a summary of phenotypic data
from persons in whom >95% of HIV-specific CD8 T cells are
PD-1.sup.+. 7 to 19 samples were analyzed for each of the indicated
phenotypic markers. The horizontal bar indicates median percentage
of tetramer.sup.+ PD-1.sup.+ cells that were positive for the
indicated marker.
[0051] FIG. 8A is a series of images of a flow cytometry experiment
showing the representative proliferation assay data from a B*4201
positive subject. After a 6-day stimulation with peptide, the
percentage of B*4201 TL9-specific CD8 T cells increased from 5.7%
to 12.4% in the presence of anti-PD-L1 blocking antibody.
[0052] FIG. 8B is a line graph depicting the summary proliferation
assay data indicating a significant increase in proliferation of
HIV-specific CD8 T cells in the presence of anti-PD-L1 blocking
antibody (n=28, p=0.0006, paired t-test).
[0053] FIG. 8C is a bar graph showing the differential effects of
PD-1/PD-L1 blockade on proliferation of HIV-specific CD8 T cells on
an individual patient basis. White bars indicate fold increase of
tetramer.sup.+ cells in the presence of peptide alone, black bars
indicate the fold increase of tetramer.sup.+ cells in the presence
of peptide plus anti-PD-L1 blocking antibody. Individuals in whom
CFSE assays were performed for more than one epitope are indicated
by asterisk, square, or triangle symbols.
SEQUENCE LISTING
[0054] The Sequence Listing is submitted as an ASCII text file
6975-6975-99667-09_Sequence_Listing, Jun. 18, 2019, 2.72 KB], which
is incorporated by reference herein.
DETAILED DESCRIPTION
[0055] The use of antibiotics and vaccines in recent decades has
significantly reduced the mortality rate due to microbial
infections. The success of antimicrobial treatment modalities,
however, has been limited by the ability of certain infectious
agents to evade the immune system of the host organism and in turn,
establish a persistent infection. For example, the immune response
that is mounted against viruses such as hepatitis and HIV is not
sufficient to clear the infectious agent, which remains in the
infected subject. In such infections, antigen specific CD8+ T cells
become functionally tolerant to the infectious agent, in a state
known as `anergy` or `exhaustion`. Anergic T cells lose their
cytotoxic activity, i.e., their ability to produce cytokines,
proliferate, and clear the infectious agent.
[0056] The present invention is based upon the surprising discovery
that T cell anergy is concomitant with an induction in PD-1
expression and that PD-1 expression correlates with certain types
of lymphoproliferative disorders. Accordingly, the invention
provides methods of increasing T-cell cytoxicity by contacting a
T-cell with an agent that reduces the expression or activity of
PD-1, PD-1 ligand (PD-L1) or PD-1 ligand 2 (PD-L2). More
specifically, the invention provides methods of treating or
preventing a persistent infection or lymphoproliferative disorders
(e.g., cancers such as angioimmunoblastic lymphoma and nodular
lymphocyte predominant Hodgkin lymphoma by administering to a
subject an agent that reduces the expression or activity of PD-1.
Reduction of PD-1, PD-L1 or PD-L2 expression or activity results in
an increase in cytotoxic T cell activity, increasing the specific
immune response to the infectious agent. The results provided
herein show that the administration of anti-programmed death
ligand-1 (PD-L1) blocking antibodies to persistently infected mice
increased the cytotoxic activity of anergic T cells. Specifically,
disruption of PD-1 signaling induced the expansion of anergic CD8+
T cells, enhanced cytokine production, and increased viral
clearance. Furthermore, CD8+ T cells generated during persistent
infections of CD4 depleted mice proliferated and regained much of
their function upon anti-PD-L1 treatment.
[0057] In order for T cells to respond to foreign proteins, two
signals must be provided by antigen-presenting cells (APCs) to
resting T lymphocytes. The first signal, which confers specificity
to the immune response, is transduced via the T cell receptor (TCR)
following recognition of foreign antigenic peptide presented in the
context of the major histocompatibility complex (MHC). The second
signal, termed costimulation, induces T cells to proliferate and
become functional. Costimulation is neither antigen-specific, nor
MHC-restricted and is provided by one or more distinct cell surface
polypeptides expressed by APCs. If T cells are only stimulated
through the T cell receptor, without receiving an additional
costimulatory signal, they become nonresponsive, anergic, or die,
resulting in downmodulation of the immune response.
[0058] The CD80 (B7-1) and CD86 (B7-2) proteins, expressed on APCs,
are critical costimulatory polypeptides. While B7-2 plays a
predominant role during primary immune responses, B7-1 is
upregulated later in the course of an immune response to prolong
primary T cell responses or costimulating secondary T cell
responses. B7 polypeptides are capable of providing costimulatory
or inhibitory signals to immune cells to promote or inhibit immune
cell responses. For example, when bound to a costimulatory
receptor, PD-L1 (B7-4) induces costimulation of immune cells or
inhibits immune cell costimulation when present in a soluble form.
When bound to an inhibitory receptor, B7-4 molecules can transmit
an inhibitory signal to an immune cell. Exemplary B7 family members
include B7-1, B7-2, B7-3 (recognized by the antibody BB-1), B7h
(PD-L1), and B7-4 and soluble fragments or derivatives thereof. B7
family members bind to one or more receptors on an immune cell,
such as CTLA4, CD28, ICOS, PD-1 and/or other receptors, and,
depending on the receptor, have the ability to transmit an
inhibitory signal or a costimulatory signal to an immune cell.
[0059] CD28 is a receptor that is constitutively expressed on
resting T cells. After signaling through the T cell receptor,
ligation of CD28 and transduction of a costimulatory signal induces
T cells to proliferate and secrete IL-2. CTLA4 (CD152), a receptor
homologous to CD28, is absent on resting T cells but its expression
is induced following T cell activation. CTLA4 plays a role in
negative regulation of T cell responses. ICOS, a polypeptide
related to CD28 and CTLA4, is involved in IL-10 production. PD-1,
the receptor to which PD-L1 and PD-L2 bind, is also rapidly induced
on the surface of T-cells. PD-1 is also expressed on the surface of
B-cells (in response to anti-IgM) and on a subset of thymocytes and
myeloid cells.
[0060] Engagement of PD-1 (for example by crosslinking or by
aggregation), leads to the transmission of an inhibitory signal in
an immune cell, resulting in a reduction of immune responses
concomitant with an increase in immune cell anergy. PD-1 family
members bind to one or more receptors, such as PD-L1 and PD-L2 on
antigen presenting cells.
[0061] PD-L1 and PD-L2, both of which are human PD-1 ligand
polypeptides, are members of the B7 family of polypeptides. Each
PD-1 ligand contains a signal sequence, an IgV domain, an IgC
domain, a transmembrane domain, and a short cytoplasmic tail. These
ligands are expressed in placenta, spleen, lymph nodes, thymus, and
heart. PD-L2 is also expressed in the pancreas, lung, and liver,
while PD-L1 is expressed in fetal liver, activated T-cells and
endothelial cells. Both PD-1 ligands are upregulated on activated
monocytes and dendritic cells.
Definitions
[0062] As used herein, by "persistent infection" is meant an
infection in which the infectious agent (e.g., virus, bacterium,
parasite, mycoplasm, or fungus) is not cleared or eliminated from
the infected host, even after the induction of an immune response.
Persistent infections may be chronic infections, latent infections,
or slow infections. While acute infections are relatively brief
(lasting a few days to a few weeks) and resolved from the body by
the immune system, persistent infections may last for months,
years, or even a lifetime. These infections may also recur
frequently over a long period of time, involving stages of silent
and productive infection without cell killing or even producing
excessive damage to the host cells. The causative infectious agents
may also be detected in the host (e.g., inside specific cells of
infected individuals) even after the immune response has resolved,
using standard techniques. Mammals are diagnosed as having a
persistent infection according to any standard method known in the
art and described, for example, in U.S. Pat. Nos. 6,368,832,
6,579,854, and 6,808,710 and U.S. Patent Application Publication
Nos. 20040137577, 20030232323, 20030166531, 20030064380,
20030044768, 20030039653, 20020164600, 20020160000, 20020110836,
20020107363, and 20020106730, all of which are hereby incorporated
by reference. For example, a subject may be diagnosed as having a
persistent Chlamydial infection following the detection of
Chlamydial species in a biological sample from this individual
using PCR analysis. Mammals need not have not been diagnosed with a
persistent infection to be treated according to this invention.
Microbial agents capable of establishing a persistent infection
include viruses (e.g., papilloma virus, hepatitis virus, human
immune deficiency virus, and herpes virus), bacteria (e.g.,
Escherichia coli and Chlamydia spp.), parasites, (e.g., Plasmodium,
Leishmania spp., Schistosoma spp., Trypanosoma spp., Toxoplasma
spp.) and fungi.
[0063] By "alleviating a symptom of a persistent infection" is
meant ameliorating any of the conditions or symptoms associated
with the persistent infection before or after it has occurred.
Alternatively, alleviating a symptom of a persistent infection may
involve reducing the infectious microbial (e.g., viral, bacterial,
fungal, mycoplasm, or parasitic) load in the subject relative to
such load in an untreated control. As compared with an equivalent
untreated control, such reduction or degree of prevention is at
least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as
measured by any standard technique. Desirably, the persistent
infection is completely cleared as detected by any standard method
known in the art, in which case the persistent infection is
considered to have been treated. A patient who is being treated for
a persistent infection is one who a medical practitioner has
diagnosed as having such a condition. Diagnosis may be by any
suitable means. Diagnosis and monitoring may involve, for example,
detecting the level of microbial load in a biological sample (e.g.,
tissue biopsy, blood test, or urine test), detecting the level of a
surrogate marker of the microbial infection in a biological sample,
detecting symptoms associated with persistent infections, or
detecting immune cells involved in the immune response typical of
persistent infections (e.g., detection of antigen specific T cells
that are anergic) A patient in whom the development of a persistent
infection is being prevented may or may not have received such a
diagnosis. One in the art will understand that these patients may
have been subjected to the same standard tests as described above
or may have been identified, without examination, as one at high
risk due to the presence of one or more risk factors (e.g., family
history or exposure to infectious agent).
[0064] As used herein, by "PD-1" is meant a polypeptide that forms
a complex with PD-L1 or PD-L2 proteins and is therefore involved in
immune responses, such as the co-stimulation of T cells. The PD-1
proteins of the invention are substantially identical to the
naturally occurring PD-1 (see, for example, Ishida et al. EMBO J.
11:3887-3895, 1992, Shinohara et al. Genomics 23:704-706, 1994; and
U.S. Pat. No. 5,698,520, incorporated by reference herein). PD-1
signaling may reduce, for example, CD8+ T cell cytoxicity by
reducing T cell proliferation, cytokine production, or viral
clearance. According to this invention, the PD-1 polypeptide
reduces CD8+ T cell cytotoxic activity by at least 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 100% below control
levels as measured by any standard method.
[0065] By a "PD-1 gene" is meant a nucleic acid that encodes a PD-1
protein.
[0066] By "PD-1 fusion gene" is meant a PD-1 promoter and/or all or
part of a PD-1 coding region operably linked to a second,
heterologous nucleic acid sequence. In preferred embodiments, the
second, heterologous nucleic acid sequence is a reporter gene, that
is, a gene whose expression may be assayed; reporter genes include,
without limitation, those encoding glucuronidase (GUS), luciferase,
chloramphenicol transacetylase (CAT), green fluorescent protein
(GFP), alkaline phosphatase, and .beta.-galactosidase.
[0067] By "reduce the expression or activity of PD-1" is meant to
reduce the level or biological activity of PD-1 relative to the
level or biological activity of PD-1 in an untreated control.
According to this invention, such level or activity is reduced by
at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or even
greater than 100%, relative to an untreated control. For example,
the biological activity of PD-1 is reduced if binding of PD-1 to
PD-L1, PD-L2, or both is reduced, thereby resulting in a reduction
in PD-1 signaling and therefore resulting in an increase in CD8+ T
cell cytotoxicity. As used herein, the term "activity" with respect
to a PD-1 polypeptide includes any activity which is inherent to
the naturally occurring PD-1 protein, such as the ability to
modulate an inhibitory signal in an activated immune cell, e.g., by
engaging a natural ligand on an antigen presenting cell. Such
modulation of an inhibitory signal in an immune cell results in
modulation of proliferation of and/or cytokine secretion by an
immune cell. PD-1 may also modulate a costimulatory signal by
competing with a costimulatory receptor for binding of a B7
molecule. Thus, the term "PD-1 activity" includes the ability of a
PD-1 polypeptide to bind its natural ligand(s), the ability to
modulate immune cell costimulatory or inhibitory signals, and the
ability to modulate the immune response. Accordingly, reducing PD-1
activity includes reducing the interaction of PD-1 to PD-L1 or
PD-L2. This can be accomplished for example by blocking PD-L1 or
PD-L2.
[0068] By "immune cell" is meant a cell of hematopoietic origin and
that plays a role in the immune response. Immune cells include
lymphocytes (e.g., B cells and T cells), natural killer cells, and
myeloid cells (e.g., monocytes, macrophages, eosinophils, mast
cells, basophils, and granulocytes).
[0069] By "T cell" is meant a CD4+ T cell or a CD8+ T cell. The
term T cell includes both TH1 cells and TH2 cells.
[0070] The term "T cell cytoxicity" includes any immune response
that is mediated by CD8+ T cell activation. Exemplary immune
responses include cytokine production, CD8+ T cell proliferation,
granzyme or perforin production, and clearance of the infectious
agent.
[0071] By "unresponsiveness" includes refractivity of immune cells
to stimulation, e.g., stimulation via an activating receptor or a
cytokine. Unresponsiveness can occur, e.g., because of exposure to
immunosuppressants or exposure to high doses of antigen. As used
herein, the term "anergy" or "tolerance" includes refractivity to
activating receptor-mediated stimulation. Such refractivity is
generally antigen-specific and persists after exposure to the
tolerizing antigen has ceased. For example, anergy in T cells (as
opposed to unresponsiveness) is characterized by lack of cytokine
production, e.g., IL-2. T cell anergy occurs when T cells are
exposed to antigen and receive a first signal (a T cell receptor or
CD-3 mediated signal) in the absence of a second signal (a
costimulatory signal). Under these conditions, re-exposure of the
cells to the same antigen (even if reexposure occurs in the
presence of a costimulatory molecule) results in failure to produce
cytokines and, thus, failure to proliferate. Anergic T cells can,
however, mount responses to unrelated antigens and can proliferate
if cultured with cytokines (e.g., IL-2). For example, T cell anergy
can also be observed by the lack of IL-2 production by T
lymphocytes as measured by ELISA or by a proliferation assay using
an indicator cell line. Alternatively, a reporter gene construct
can be used. For example, anergic T cells fail to initiate IL-2
gene transcription induced by a heterologous promoter under the
control of the 5' IL-2 gene enhancer or by a multimer of the AP1
sequence that can be found within the enhancer (Kang et al. Science
257:1134, 1992). Anergic antigen specific T cells may have a
reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or even 100% in cytotoxic activity relative a corresponding
control antigen specific T cell.
[0072] By "purified antibody" is meant antibody which is at least
60%, by weight, free from proteins and naturally occurring organic
molecules with which it is naturally associated. Preferably, the
preparation is at least 75%, more preferably 90%, and most
preferably at least 99%, by weight, antibody, e.g., a PD-1, PD-L1,
or PD-L2 specific antibody. A purified antibody may be obtained,
for example, by affinity chromatography using
recombinantly-produced protein or conserved motif peptides and
standard techniques.
[0073] By "specifically binds" is meant an antibody that recognizes
and binds an antigen such as a PD-1, PD-L1, or PD-L2 polypeptide
but that does not substantially recognize and bind other
non-antigen molecules in a sample, e.g., a biological sample, that
naturally includes protein. A preferred antibody binds to the PD-1,
PD-L1, or PD-L2 polypeptides disclosed in U.S. Pat. No. 6,808,710
and U.S. Patent Application Publication Nos. 20040137577,
20030232323, 20030166531, 20030064380, 20030044768, 20030039653,
20020164600, 20020160000, 20020110836, 20020107363, and
20020106730, all of which are hereby incorporated by reference.
[0074] By "neutralizing antibodies" is meant antibodies that
interfere with any of the biological activities of a PD-1
polypeptide, particularly the ability of a PD-1 polypeptide to
reduce an immune response such as the cytotoxicity of T cells. The
neutralizing antibody may reduce the ability of a PD-1 polypeptide
to reduce an immune response by, preferably 50%, more preferably by
70%, and most preferably by 90% or more. Any standard assay to
measure immune responses, including those described herein, may be
used to assess potentially neutralizing antibodies.
[0075] By "substantially identical," when referring to a protein or
polypeptide, is meant a protein or polypeptide exhibiting at least
75%, but preferably 85%, more preferably 90%, most preferably 95%,
or even 99% identity to a reference amino acid sequence. For
proteins or polypeptides, the length of comparison sequences will
generally be at least 20 amino acids, preferably at least 30 amino
acids, more preferably at least 40 amino acids, and most preferably
50 amino acids or the full length protein or polypeptide. Nucleic
acids that encode such "substantially identical" proteins or
polypeptides constitute an example of "substantially identical"
nucleic acids; it is recognized that the nucleic acids include any
sequence, due to the degeneracy of the genetic code, that encodes
those proteins or polypeptides. In addition, a "substantially
identical" nucleic acid sequence also includes a polynucleotide
that hybridizes to a reference nucleic acid molecule under high
stringency conditions.
[0076] By "high stringency conditions" is meant any set of
conditions that are characterized by high temperature and low ionic
strength and allow hybridization comparable with those resulting
from the use of a DNA probe of at least 40 nucleotides in length,
in a buffer containing 0.5 M NaHPO.sub.4, pH 7.2, 7% SDS, 1 mM
EDTA, and 1% BSA (Fraction V), at a temperature of 65.degree. C.,
or a buffer containing 48% formamide, 4.8.times.SSC, 0.2 M Tris-C1,
pH 7.6, 1.times.Denhardt's solution, 10% dextran sulfate, and 0.1%
SDS, at a temperature of 42.degree. C. Other conditions for high
stringency hybridization, such as for PCR, Northern, Southern, or
in situ hybridization, DNA sequencing, etc., are well known by
those skilled in the art of molecular biology. See, e.g., F.
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
& Sons, New York, N.Y., 1998, hereby incorporated by
reference.
[0077] By "substantially pure" is meant a nucleic acid,
polypeptide, or other molecule that has been separated from the
components that naturally accompany it. Typically, the polypeptide
is substantially pure when it is at least 60%, 70%, 80%, 90%, 95%,
or even 99%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. For example, a substantially pure polypeptide may be
obtained by extraction from a natural source, by expression of a
recombinant nucleic acid in a cell that does not normally express
that protein, or by chemical synthesis.
[0078] The term "isolated DNA" is meant DNA that is free of the
genes which, in the naturally occurring genome of the organism from
which the given DNA is derived, flank the DNA. Thus, the term
"isolated DNA" encompasses, for example, cDNA, cloned genomic DNA,
and synthetic DNA.
[0079] By "an effective amount" is meant an amount of a compound,
alone or in a combination, required to reduce or prevent
hypertension or to treat or prevent a chronic infection in a
mammal. The effective amount of active compound(s) varies depending
upon the route of administration, age, body weight, and general
health of the subject. Ultimately, the attending physician or
veterinarian will decide the appropriate amount and dosage
regimen.
[0080] By a "candidate compound" is meant a chemical, be it
naturally-occurring or artificially-derived. Candidate compounds
may include, for example, peptides, polypeptides, synthetic organic
molecules, naturally occurring organic molecules, nucleic acid
molecules, peptide nucleic acid molecules, and components and
derivatives thereof. For example, a useful candidate compound
according to the present invention reduces binding of PD-1 to
PD-L1, PD-L2, or both.
[0081] The term "pharmaceutical composition" is meant any
composition, which contains at least one therapeutically or
biologically active agent and is suitable for administration to the
patient. Any of these formulations can be prepared by well-known
and accepted methods of the art. See, for example, Remington: The
Science and Practice of Pharmacy, 20.sup.th edition, (ed. A R
Gennaro), Mack Publishing Co., Easton, Pa., 2000.
Methods of Treating
[0082] T-cell cytotoxicity is increased by contacting a T-cell with
a compound that reduces the expression or activity of PD-1. The
T-cell is a naive T-cell, memory T-cell or activated T-cell.
Alternatively, the T-cell is an antigen specific T-cell. The
antigen specific T cells is anergic or tolerant to the infectious
agent. T-cell cytotoxicity is characterized by an increase in cell
proliferation and cytokine release.
[0083] The methods are useful to alleviate the symptoms of a
variety of infections and cancers. An infection or cancer is
treated, prevented or a symptom is alleviated by administering to a
subject a PD-1 inhibitor. The subject is a mammal such as human, a
primate, mouse, rat, dog, cat, cow, horse, and pig. The subject is
suffering from or at risk of developing infection. A subject
suffering from or at risk of developing infection is by standard
methods suitable for the particular infection.
[0084] The infection, e.g., bacterial, viral, fungal, mycoplasm, or
parasitic is a persistent infection. Persistent infections, in
contrast to acute infections are not effectively cleared by the
induction of a host immune response. The infectious agent and the
immune response reach equilibrium such that the infected subject
remains infectious over a long period of time without necessarily
expressing symptoms. Persistent infections include for example,
latent, chronic and slow infections.
[0085] In a chronic infection, the infectious agent can be detected
in the body at all times. However, the signs and symptoms of the
disease may be present or absent for an extended period of time.
Examples of chronic infection include hepatitis B (caused by HBV)
and hepatitis C (caused by HCV) adenovirus, cytomegalovirus,
Epstein-Barr virus, herpes simplex virus 1, herpes simplex virus 2,
human herpesvirus 6, varicella-zoster virus, hepatitis B virus,
hepatitis D virus, papilloma virus, parvovirus B19, polyomavirus
BK, polyomavirus JC, measles virus, rubella virus, human
immunodeficiency virus (HIV), human T cell leukemia virus I, and
human T cell leukemia virus II. Parasitic persistent infections may
arise as a result of infection by Leishmania, Toxoplasma,
Trypanosoma, Plasmodium, Schistosoma, and Encephalitozoon.
[0086] In a latent infection, the infectious agent (e.g., virus) is
seemingly inactive and dormant such that the subject does always
exhibit signs or symptoms. In a latent viral infection, the virus
remains in equilibrium with the host for long periods of time
before symptoms again appear; however, the actual viruses cannot be
detected until reactivation of the disease occurs. Examples of
latent infections include infections caused by HSV-1 (fever
blisters), HSV-2 (genital herpes), and VZV
(chickenpox-shingles).
[0087] In a slow infection, the infectious agents gradually
increase in number over a very long period of time during which no
significant signs or symptoms are observed. Examples of slow
infections include AIDS (caused by HIV-1 and HIV-2), lentiviruses
that cause tumors in animals, and prions.
[0088] In addition, persistent infections often arise as late
complications of acute infections. For example, subacute sclerosing
panencephalitis (SSPE) can occur following an acute measles
infection or regrossive encephalitis can occur as a result of a
rubella infection.
[0089] Cancers include for example angioimmunoblastic lymphoma or
nodular lymphocyte predominant Hodgkin lymphoma.
[0090] Angioimmunoblastic lymphoma (AIL) is an aggressive (rapidly
progressing) type of T-cell non-Hodgkin lymphoma marked by enlarged
lymph nodes and hypergammaglobulinemia (increased antibodies in the
blood). Other symptoms may include a skin rash, fever, weight loss,
positive Coomb's test or night sweats. This malignancy usually
occurs in adults. Patients are usually aged 40-90 years (median
around 65) and are more often male. As AIL progresses,
hepatosplenomegaly, hemolytic anemia, and polyclonal
hypergammaglobulinemia may develop. The skin is involved in
approximately 40-50% of patients.
[0091] Nodular lymphocyte predominant Hodgkin lymphoma is a B cell
neoplasm that appears to be derived from germinal center B cells
with mutated, non-functional immunoglobulin genes. Similar to
angioimmunoblastic lymphoma, neoplastic cells are associated with a
meshwork of follicular dendritic cells. PD-1 expression is seen in
T cells closely associated with neoplastic CD20+ cell in nodular
lymphocyte predominant Hodgkin lymphoma, in a pattern similar to
that seen for CD57+ T cells. CD57 has been identified as another
marker of germinal center-associated T cells, along with CXCR5,
findings which support the conclusion that the neoplastic cells in
nodular lymphocyte predominant Hodgkin lymphoma have a close
association with germinal center-associated T cells.
[0092] An inhibitor of PD-1 is any agent having the ability to
reduce the expression or the activity of PD-1, PD-L1 or PD-2 in a
cell. PD-1 expression or activity is reduced by at least 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to such
expression or activity in a control cell. The control cell is a
cell that has not been treated with the PD-1 inhibitor. PD-1
expression or activity is determined by any standard method in the
art, including those described herein. Optionally, the PD-1
inhibitor inhibits or reduces binding of PD-1 to PD-L1, PD-L2, or
both. PD-1 inhibitors include polypeptides, polynucleotides, small
molecule antagonists, or siRNA.
[0093] A PD-1 inhibitor polypeptide includes, for example, an
antibody or fragment thereof that reduces PD-1 expression or
signaling. Exemplary antibodies include anti-PD-1 antibodies,
anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4
antibodies, anti-BTLA antibodies, anti-CD28 antibodies, anti-ICOS
antibodies, anti-ICOS-L antibodies, an anti-B7-1 antibody, an
anti-B7-2 antibody, anti-B7-H3 antibodies, or anti-B7-H4
antibodies.
[0094] Alternatively, the PD-1 inhibitor is a dominant negative
protein or a nucleic acid encoding a dominant negative protein that
interferes with the biological activity of PD-1 (i.e. binding of
PD-1 to PD-L1, PD-L2, or both). A dominant negative protein is any
amino acid molecule having a sequence that has at least 50%, 70%,
80%, 90%, 95%, or even 99% sequence identity to at least 10, 20,
35, 50, 100, or more than 150 amino acids of the wild type protein
to which the dominant negative protein corresponds. For example, a
dominant-negative PD-1 has mutation such that it no longer able to
binds PD-L1.
[0095] The dominant negative protein may be administered as an
expression vector. The expression vector may be a non-viral vector
or a viral vector (e.g., retrovirus, recombinant adeno-associated
virus, or a recombinant adenoviral vector). Alternatively, the
dominant negative protein may be directly administered as a
recombinant protein systemically or to the infected area using, for
example, microinjection techniques.
[0096] Small molecules includes, but are not limited to, peptides,
peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,
polynucleotides, polynucleotide analogs, nucleotides, nucleotide
analogs, organic and inorganic compounds (including heterorganic
and organomettallic compounds) having a molecular weight less than
about 5,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 2,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 1,000
grams per mole, organic or inorganic compounds having a molecular
weight less than about 500 grams per mole, and salts, esters, and
other pharmaceutically acceptable forms of such compounds.
[0097] The PD-1 inhibitor is an antisense molecule, an RNA
interference (siRNA) molecule, or a small molecule antagonist that
targets PD-1 expression or activity. By the term "siRNA" is meant a
double stranded RNA molecule which prevents translation of a target
mRNA. Standard techniques of introducing siRNA into a cell are
used, including those in which DNA is a template from which an
siRNA RNA is transcribed. The siRNA includes a sense PD-1, PD-L1 or
PD-L2 nucleic acid sequence, an anti-sense PD-1, PD-L1 or PD-L2
nucleic acid sequence or both. Optionally, the siRNA is constructed
such that a single transcript has both the sense and complementary
antisense sequences from the target gene, e.g., a hairpin. Binding
of the siRNA to an PD-1, PD-L1 or PD-L2 transcript in the target
cell results in a reduction in PD-1, PD-L1 or PD-L2production by
the cell. The length of the oligonucleotide is at least 10
nucleotides and may be as long as the naturally-occurring PD-1,
PD-L1 or PD-L2 transcript. Preferably, the oligonucleotide is 19-25
nucleotides in length. Most preferably, the oligonucleotide is less
than 75, 50, 25 nucleotides in length.
[0098] Other suitable PD-1 inhibitors are described in, for
example, in U.S. Pat. No. 6,808,710 and U.S. Patent Application
Publication Nos. 20040137577, 20030232323, 20030166531,
20030064380, 20030044768, 20030039653, 20020164600, 20020160000,
20020110836, 20020107363, and 20020106730, all of which are hereby
incorporated by reference.
[0099] The preferred dose of the PD-1 inhibitor is a biologically
active dose. A biologically active dose is a dose that will induce
an increase in CD8+ T cell cytotoxic activity the increase in the
immune response specific to the infectious agent. Desirably, the
PD-1 inhibitor has the ability to reduce the expression or activity
of PD-1 in antigen specific immune cells (e.g., T cells such as
CD8+ T cells) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, or more than 100% below untreated control levels. The
levels or activity of PD-1 in immune cells is measured by any
method known in the art, including, for example, Western blot
analysis, immunohistochemistry, ELISA, and Northern Blot analysis.
Alternatively, the biological activity of PD-1 is measured by
assessing binding of PD-1 to PD-L1, PD-L2, or both. The biological
activity of PD-1 is determined according to its ability to increase
CD8+ T cell cytotoxicity including, for example, cytokine
production, clearance of the infectious agent, and proliferation of
antigen specific CD8+ T cells. Preferably, the agent that reduces
the expression or activity of PD-1 can increase the immune response
specific to the infectious agent by at least 5%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, or more than 100% above untreated
control levels. The agent of the present invention is therefore any
agent having any one or more of these activities. Although the
agent of the invention is preferably expressed in CD8+ T cells, it
is understood that any cell that can influence the immune response
to persistent infections is also amenable to the methods of the
invention and include, for example, B cells.
[0100] Optionally, the subject is administered one or more
additional therapeutic agents. Additional therapeutic agents
include, for example, antiviral compounds (e.g., vidarabine,
acyclovir, gancyclovir, valgancyclovir, nucleoside-analog reverse
transcriptase inhibitor (NRTI) (e.g., AZT (Zidovudine), ddI
(Didanosine), ddC (Zalcitabine), d4T (Stavudine), or 3TC
(Lamivudine)), non-nucleoside reverse transcriptase inhibitor
(NNRTI) (e.g., (nevirapine or delavirdine), protease inhibitor
(saquinavir, ritonavir, indinavir, or nelfinavir), ribavirin, or
interferon), antibacterial compounds, antifungal compounds,
antiparasitic compounds, anti-inflammatory compounds,
anti-neoplastic agent or analgesics.
[0101] The additional therapeutic agent is administered prior to,
concomitantly, or subsequent to administration of the PD-1
inhibitor. For example, the PD-1 inhibitor and the additional agent
are administered in separate formulations within at least 1, 2, 4,
6, 10, 12, 18, or more than 24 hours apart. Optionally, the
additional agent is formulated together with the PD-1 inhibitor.
When the additional agent is present in a different composition,
different routes of administration may be used. The agent is
administered at doses known to be effective for such agent for
treating, reducing, or preventing an infection.
[0102] Concentrations of the PD-1 inhibitor and the additional
agent depends upon different factors, including means of
administration, target site, physiological state of the mammal, and
other medication administered. Thus treatment dosages may be
titrated to optimize safety and efficacy and is within the skill of
an artisan. Determination of the proper dosage and administration
regime for a particular situation is within the skill of the
art.
[0103] Optionally, the subject is further administered a vaccine
that elicits a protective immune response against the infectious
agent that causes a persistent infection. For example, the subject
receives a vaccine that elicits an immune response against human
immunodeficiency virus (HIV), tuberculosis, influenza, or hepatitis
C. Exemplary vaccines are described, for example, in Berzofsky et
al. (J. Clin. Invest. 114:456-462, 2004). If desired, the vaccine
is administered with a prime-booster shot or with adjuvants.
[0104] PD-1 inhibitors are administered in an amount sufficient to
increase T cell, e.g., CD8+ T cell, cytotoxicity. An increase in
T-cell cytotoxicity results in an increased immune response and a
reduction in the persistent infection. An increased immune response
is measured, for example, by an increase in immune cell
proliferation, e.g., T-cell or B cell, an increase in cytokine
production, and an increase in the clearance of an infectious
agent. Such reduction includes the alleviation of one or more of
symptoms associated with the persistent infection. Administration
of the PD-1 inhibitor reduces the persistent infection or
alleviates one or more symptoms associated with the persistent
infection by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
or 100% as compared to an untreated subject.
[0105] Treatment is efficacious if the treatment leads to clinical
benefit such as, a reduction of the load of the infectious agent in
the subject. When treatment is applied prophylactically,
"efficacious" means that the treatment retards or prevents an
infection from forming. Efficacy may be determined using any known
method for diagnosing or treating the particular infection.
Therapeutic Administration
[0106] The invention includes administering to a subject a
composition that includes a compound that reduces PD-1 expression
or activity (referred to herein as an "PD-1 inhibitor" or
"therapeutic compound").
[0107] An effective amount of a therapeutic compound is preferably
from about 0.1 mg/kg to about 150 mg/kg. Effective doses vary, as
recognized by those skilled in the art, depending on route of
administration, excipient usage, and coadministration with other
therapeutic treatments including use of other anti-infection agents
or therapeutic agents for treating, preventing or alleviating a
symptom of a particular infection or cancer. A therapeutic regimen
is carried out by identifying a mammal, e.g., a human patient
suffering from (or at risk of developing) an infection or cancer,
using standard methods.
[0108] The pharmaceutical compound is administered to such an
individual using methods known in the art. Preferably, the compound
is administered orally, rectally, nasally, topically or
parenterally, e.g., subcutaneously, intraperitoneally,
intramuscularly, and intravenously. The compound is administered
prophylactically, or after the detection of an infection. The
compound is optionally formulated as a component of a cocktail of
therapeutic drugs to treat infection. Examples of formulations
suitable for parenteral administration include aqueous solutions of
the active agent in an isotonic saline solution, a 5% glucose
solution, or another standard pharmaceutically acceptable
excipient. Standard solubilizing agents such as PVP or
cyclodextrins are also utilized as pharmaceutical excipients for
delivery of the therapeutic compounds.
[0109] The therapeutic compounds described herein are formulated
into compositions for other routes of administration utilizing
conventional methods. For example, PD-1 inhibitor is formulated in
a capsule or a tablet for oral administration. Capsules may contain
any standard pharmaceutically acceptable materials such as gelatin
or cellulose. Tablets may be formulated in accordance with
conventional procedures by compressing mixtures of a therapeutic
compound with a solid carrier and a lubricant. Examples of solid
carriers include starch and sugar bentonite. The compound is
administered in the form of a hard shell tablet or a capsule
containing a binder, e.g., lactose or mannitol, a conventional
filler, and a tableting agent. Other formulations include an
ointment, suppository, paste, spray, patch, cream, gel, resorbable
sponge, or foam. Such formulations are produced using methods well
known in the art.
[0110] Where the therapeutic compound is a nucleic acid encoding a
protein, the Therapeutic nucleic acid is administered in vivo to
promote expression of its encoded protein, by constructing it as
part of an appropriate nucleic acid expression vector and
administering it so that it becomes intracellular (e.g., by use of
a retroviral vector, by direct injection, by use of microparticle
bombardment, by coating with lipids or cell-surface receptors or
transfecting agents, or by administering it in linkage to a
homeobox-like peptide which is known to enter the nucleus (See,
e.g., Joliot, et al., 1991. Proc Natl Acad Sci USA 88:1864-1868),
and the like. Alternatively, a nucleic acid therapeutic is
introduced intracellularly and incorporated within host cell DNA
for expression, by homologous recombination or remain episomal.
[0111] For local administration of DNA, standard gene therapy
vectors used. Such vectors include viral vectors, including those
derived from replication-defective hepatitis viruses (e.g., HBV and
HCV), retroviruses (see, e.g., WO 89/07136; Rosenberg et al., 1990,
N. Eng. J. Med. 323(9):570-578), adenovirus (see, e.g., Morsey et
al., 1993, J. Cell. Biochem., Supp. 17E,), adeno-associated virus
(Kotin et al., 1990, Proc. Natl. Acad. Sci. USA 87:2211-2215,),
replication defective herpes simplex viruses (HSV; Lu et al., 1992,
Abstract, page 66, Abstracts of the Meeting on Gene Therapy,
September 22-26, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.), and any modified versions of these vectors. The invention
may utilize any other delivery system which accomplishes in vivo
transfer of nucleic acids into eucaryotic cells. For example, the
nucleic acids may be packaged into liposomes, e.g., cationic
liposomes (Lipofectin), receptor-mediated delivery systems,
non-viral nucleic acid-based vectors, erytbrocyte ghosts, or
microspheres (e.g., microparticles; see, e.g., U.S. Pat. Nos.
4,789,734; 4,925,673; 3,625,214; Gregoriadis, 1979, Drug Carriers
in Biology and Medicine, pp. 287-341 (Academic Press,). Naked DNA
may also be administered.
[0112] DNA for gene therapy can be administered to patients
parenterally, e.g., intravenously, subcutaneously, intramuscularly,
and intraperitoneally. DNA or an inducing agent is administered in
a pharmaceutically acceptable carrier, i.e., a biologically
compatible vehicle which is suitable for administration to an
animal e.g., physiological saline. A therapeutically effective
amount is an amount which is capable of producing a medically
desirable result, e.g., an decrease of a PD-1 gene product in a
treated animal. Such an amount can be determined by one of ordinary
skill in the art. As is well known in the medical arts, dosage for
any given patient depends upon many factors, including the
patient's size, body surface area, age, the particular compound to
be administered, sex, time and route of administration, general
health, and other drugs being administered concurrently. Dosages
may vary, but a preferred dosage for intravenous administration of
DNA is approximately 10.sup.6 to 10.sup.22 copies of the DNA
molecule. Typically, plasmids are administered to a mammal in an
amount of about 1 nanogram to about 5000 micrograms of DNA.
Desirably, compositions contain about 5 nanograms to 1000
micrograms of DNA, 10 nanograms to 800 micrograms of DNA, 0.1
micrograms to 500 micrograms of DNA, 1 microgram to 350 micrograms
of DNA, 25 micrograms to 250 micrograms of DNA, or 100 micrograms
to 200 micrograms of DNA. Alternatively, administration of
recombinant adenoviral vectors encoding the PD-1 inhibitor into a
mammal may be administered at a concentration of at least 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, or 10.sup.11
plaque forming unit (pfu).
[0113] PD-1 gene products are administered to the patient
intravenously in a pharmaceutically acceptable carrier such as
physiological saline. Standard methods for intracellular delivery
of peptides can be used, e.g. packaged in liposomes. Such methods
are well known to those of ordinary skill in the art. It is
expected that an intravenous dosage of approximately 1 to 100 moles
of the polypeptide of the invention would be administered per kg of
body weight per day. The compositions of the invention are useful
for parenteral administration, such as intravenous, subcutaneous,
intramuscular, and intraperitoneal.
[0114] PD-1 inhibitors are effective upon direct contact of the
compound with the affected tissue. Accordingly, the compound may be
administered topically. Alternatively, PD-1 inhibitors may be
administered systemically. Additionally, compounds may be
administered by implanting (either directly into an organ (e.g.,
intestine or liver) or subcutaneously) a solid or resorbable matrix
which slowly releases the compound into adjacent and surrounding
tissues of the subject. For example, for the treatment of
gastrointestinal infection, the compound may be administered
systemically (e.g., intravenously, rectally or orally) or locally
(e.g., directly into gastric tissue). Alternatively, a PD-1
inhibitor-impregnated wafer or resorbable sponge is placed in
direct contact with gastric tissue. The PD-1 inhibitor is slowly
released in vivo by diffusion of the drug from the wafer and
erosion of the polymer matrix. As another example, infection of the
liver (i.e., hepatitis) is treated by infusing into the liver
vasculature a solution containing the PD-1 inhibitor.
[0115] For the treatment of neurological infections, the PD-1
inhibitor may be administered intravenously or intrathecally (i.e.,
by direct infusion into the cerebrospinal fluid). For local
administration, a compound-impregnated wafer or resorbable sponge
is placed in direct contact with CNS tissue. The compound or
mixture of compounds is slowly released in vivo by diffusion of the
drug from the wafer and erosion of the polymer matrix.
Alternatively, the compound is infused into the brain or
cerebrospinal fluid using standard methods. For example, a burr
hole ring with a catheter for use as an injection port is
positioned to engage the skull at a burr hole drilled into the
skull. A fluid reservoir connected to the catheter is accessed by a
needle or stylet inserted through a septum positioned over the top
of the burr hole ring. A catheter assembly (described, for example,
in U.S. Pat. No. 5,954,687) provides a fluid flow path suitable for
the transfer of fluids to or from selected location at, near or
within the brain to allow administration of the drug over a period
of time.
[0116] For cardiac infections, the compound may be delivered, for
example, to the cardiac tissue (i.e., myocardium, pericardium, or
endocardium) by direct intracoronary injection through the chest
wall or using standard percutaneous catheter based methods under
fluoroscopic guidance. Thus, the inhibitor may be directly injected
into tissue or may be infused from a stent or catheter which is
inserted into a bodily lumen. Any variety of coronary catheter or
perfusion catheter may be used to administer the compound.
Alternatively, the compound is coated or impregnated on a stent
that is placed in a coronary vessel.
[0117] Pulmonary infections may be treated, for example, by
administering the compound by inhalation. The compounds are
delivered in the form of an aerosol spray from a pressured
container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide or a nebulizer.
[0118] One in the art will understand that the patients treated
according to the invention may have been subjected to the same
tests to diagnose a persistently infected subject or may have been
identified, without examination, as one at high risk due to the
presence of one or more risk factors (e.g., exposure to infectious
agent, exposure to infected subject, genetic predisposition, or
having a pathological condition predisposing to secondary
infections). Reduction of persistent infection symptoms or damage
may also include, but are not limited to, alleviation of symptoms,
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression, and
amelioration or palliation of the disease state. Treatment may
occur at home with close supervision by the health care provider,
or may occur in a health care facility.
Methods to Measure Immune Response
[0119] Methods for measuring the immune response following
treatment according to the present invention are well known in the
art. The activity of T cells may be assessed, for example, by
assays that detect cytokine production, assays measuring T cell
proliferation, assays that measure the clearance of the microbial
agent, and assays that measure CD8+ T cell cytotoxicity. These
assays are described, for example, in U.S. Pat. No. 6,808,710 and
U.S. Patent Application Publication Nos. 20040137577, 20030232323,
20030166531, 20030064380, 20030044768, 20030039653, 20020164600,
20020160000, 20020110836, 20020107363, and 20020106730, all of
which are hereby incorporated by reference.
[0120] Optionally, the ability of a PD-1 inhibitor to increase CD8+
T cell cytotoxicity is assessed by assays that measure the
proliferation of CD8+ T cells (e.g., thymidine incorporation, BrdU
assays, and staining with cell cycle markers (e.g., Ki67 and CFSE),
described, for example, by Dong et al (Nature 5:1365-1369, 1999).
In one example, T-cell proliferation is monitored by culturing the
purified T-cells expressing PD-1 with a PD-1 inhibitor, a primary
activation signal as described above, and .sup.3H-thymidine. The
level of T-cell proliferation is determined by measuring thymidine
incorporation.
[0121] CD8+ T cell cytotoxicity is also assessed by lysis assays
(e.g., 51Cr release assays or assays detecting the release of
perforin or granzyme), assays that detect caspase activation, or
assays that measure the clearance of the microbial agent from the
infected subject. For example, the viral load in a biological
sample from the infected subject (e.g., serum, spleen, liver, lung,
or the tissue to which the virus is tropic) may be measured before
and after treatment.
[0122] The production of cytokines such as IFN.gamma., TNF-.alpha.,
and IL-2 may also be measured. For example, purified T-cells are
cultured in the presence of the PD-1 inhibitor protein and a
primary activation signal. The level of various cytokines in the
supernatant can be determined by sandwich enzyme-linked
immunosorbent assays or other conventional assays described, for
example, in Dong et al (Nature 5:1365-1369, 1999).
[0123] If desired, the efficacy of the PD-1 inhibitor is assessed
by its ability to induce co-stimulation of T cells. For example, a
method for in vitro T-cell co-stimulation involves providing
purified T-cells that express PD-1 with a first or primary
activation signal by anti-CD3 monoclonal antibody or phorbol ester,
or by antigen in association with class II MHC. The ability of a
candidate compound agent to reduce PD-1 expression or activity and
therefore provide the secondary or co-stimulatory signal necessary
to modulate immune function, to these T-cells can then be assayed
by any one of the several conventional assays well known in the
art.
[0124] A B cell response is assessed by an antigen specific ELISA
(e.g., LCMV, HIV, tuberculosis, or malaria), plasma cell ELISPOT,
memory B-cell assay, phenotyping of B cell, and analysis of
germinal centers by immunohistochemistry.
Screening Assays
[0125] The present invention provides screening methods to identify
compounds that can inhibit the expression or activity of PD-1.
Useful compounds include any agent that inhibits the biological
activity or reduces the cellular level of PD-1. For example,
candidate compounds may reduce binding of PD-1 to PD-L1, PD-L2, or
both. Using such agents as lead compounds, for example, the present
screening methods also allow the identification of further novel,
specific inhibitors of PD-1 that function to treat, reduce, or
prevent persistent infections, or alternatively, that alleviate one
or more symptoms associated with such infections. The method of
screening may involve high-throughput techniques.
[0126] By a "candidate compound" is meant a chemical, be it
naturally-occurring or artificially-derived. Candidate compounds
may include, for example, peptides, polypeptides, synthetic organic
molecules, naturally occurring organic molecules, nucleic acid
molecules, peptide nucleic acid molecules, and components and
derivatives thereof. For example, a useful candidate compound
according to the present invention reduces binding of PD-1 to
PD-L1, PD-L2, or both.
[0127] A number of methods are available for carrying out such
screening assays. According to one approach, candidate compounds
are added at varying concentrations to the culture medium of cells
expressing PD-1. By a "PD-1 gene" is meant a nucleic acid that
encodes a PD-1 protein. By "PD-1 fusion gene" is meant a PD-1
promoter and/or all or part of a PD-1 coding region operably linked
to a second, heterologous nucleic acid sequence. In preferred
embodiments, the second, heterologous nucleic acid sequence is a
reporter gene, that is, a gene whose expression may be assayed;
reporter genes include, without limitation, those encoding
glucuronidase (GUS), luciferase, chloramphenicol transacetylase
(CAT), green fluorescent protein (GFP), alkaline phosphatase, and
beta-galactosidase.
[0128] Gene expression of PD-1 is then measured, for example, by
standard Northern blot analysis (Ausubel et al., supra), using any
appropriate fragment prepared from the nucleic acid molecule of
PD-1 as a hybridization probe or by real time PCR with appropriate
primers. The level of gene expression in the presence of the
candidate compound is compared to the level measured in a control
culture medium lacking the candidate molecule. If desired, the
effect of candidate compounds may, in the alternative, be measured
at the level of PD-1 polypeptide using the same general approach
and standard immunological techniques, such as Western blotting or
immunoprecipitation with an antibody specific to PD-1 for example.
For example, immunoassays may be used to detect or monitor the
level of PD-1. Polyclonal or monoclonal antibodies which are
capable of binding to PD-1 may be used in any standard immunoassay
format (e.g., ELISA or RIA assay) to measure the levels of PD-1.
PD-1 can also be measured using mass spectroscopy, high performance
liquid chromatography, spectrophotometric or fluorometric
techniques, or combinations thereof.
[0129] Alternatively, the screening methods of the invention may be
used to identify candidate compounds that decrease the biological
activity of PD-1 by reducing binding of PD-1 to PD-L1, PD-L2, or
both by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
or 100% relative to an untreated control. For example, a candidate
compound may be tested for its ability to decrease PD-1 activity in
cells that naturally express PD-1, after transfection with cDNA for
PD-1, or in cell-free solutions containing PD-1, as described
further below. The effect of a candidate compound on the binding or
activation of PD-1 can be tested by radioactive and non-radiaoctive
binding assays, competition assays, and receptor signaling
assays.
[0130] As a specific example, mammalian cells (e.g., rodent cells)
that express a nucleic acid encoding PD-1 are cultured in the
presence of a candidate compound (e.g., a peptide, polypeptide,
synthetic organic molecule, naturally occurring organic molecule,
nucleic acid molecule, or component thereof). Cells may either
endogenously express PD-1 or may alternatively be genetically
engineered by any standard technique known in the art (e.g.,
transfection and viral infection) to overexpress PD-1. The
expression level of PD-1 is measured in these cells by means of
Western blot analysis and subsequently compared to the level of
expression of the same protein in control cells that have not been
contacted by the candidate compound. A compound which promotes a
decrease in the level of PD-1 activity as a result of reducing its
synthesis or biological activity is considered useful in the
invention.
[0131] In one particular example, a compound that interferes with
PD-1 binding to PD-L, PD-L2, or both (thereby reducing the
biological activity of PD-1), leading to an increase in an immune
response, is useful according to the present invention. Given its
ability to decrease the biological activity of PD-1, such a
molecule may be used, for example, as a therapeutic agent to treat,
reduce, or prevent a persistent infection, or alternatively, to
alleviate one or more symptoms associated with such infections. As
a specific example, a candidate compound may be contacted with two
proteins, the first protein being a polypeptide substantially
identical to PD-1 and the second protein being either PD-L1 or
PD-L2 (i.e., a protein that binds the PD-1 polypeptide under
conditions that allow binding and that results in a reduced immune
response). According to this particular screening method, the
interaction between these two proteins is measured following the
addition of a candidate compound. A decrease in the binding PD-1 to
the second polypeptide following the addition of the candidate
compound (relative to such binding in the absence of the compound)
identifies the candidate compound as having the ability to inhibit
the interaction between the two proteins. Ultimately, the screening
assay of the invention may be carried out, for example, in a
cell-free system or using a yeast two-hybrid system. If desired,
one of the proteins or the candidate compound may be immobilized on
a support as described above or may have a detectable group.
[0132] Alternatively, or in addition, candidate compounds may be
screened for those which specifically bind to and thereby inhibit
PD-1. The efficacy of such a candidate compound is dependent upon
its ability to interact with PD-1. Such an interaction can be
readily assayed using any number of standard binding techniques and
functional assays (e.g., those described in Ausubel et al., supra).
For example, a candidate compound may be tested in vitro for
interaction and binding with PD-1 and its ability to modulate
immune responses may be assayed by any standard assays (e.g., those
described herein).
[0133] For example, a candidate compound that binds to PD-1 may be
identified using a chromatography-based technique. For example, a
recombinant PD-1 may be purified by standard techniques from cells
engineered to express PD-1 (e.g., those described above) and may be
immobilized on a column. Alternatively, the naturally-occurring
PD-1 may be immobilized on a column. A solution of candidate
compounds is then passed through the column, and a compound
specific for PD-1 is identified on the basis of its ability to bind
to PD-1 and be immobilized on the column. To isolate the compound,
the column is washed to remove non-specifically bound molecules,
and the compound of interest is then released from the column and
collected. Compounds isolated by this method (or any other
appropriate method) may, if desired, be further purified (e.g., by
high performance liquid chromatography).
[0134] Screening for new inhibitors and optimization of lead
compounds may be assessed, for example, by assessing their ability
to modulate cytotoxic T cell activity or the immune response using
standard techniques. In addition, these candidate compounds may be
tested for their ability to function as anti-microbial agents
(e.g., as described herein). Compounds isolated by this approach
may also be used, for example, as therapeutics to treat, reduce, or
prevent persistent infections, or alternatively, to alleviate one
or more symptoms associated with such infections. Compounds which
are identified as binding to PD-1 with an affinity constant less
than or equal to 10 mM are considered particularly useful in the
invention.
[0135] Potential therapeutic agents include organic molecules,
peptides, peptide mimetics, polypeptides, and antibodies that bind
to a nucleic acid sequence or polypeptide that encodes PD-1 and
thereby inhibit or extinguish their activity. Potential
anti-microbial agents also include small molecules that bind to and
occupy the binding site of such polypeptides thereby preventing
binding to cellular binding molecules, such that normal biological
activity is prevented. Other potential anti-microbial agents
include antisense molecules.
Diagnostic and Prognostic Methods
[0136] Cancer, e.g. angioimmunoblastic T cell lymphoma or Nodular
Lymphocyte Predominant Hodgkin's Lymphoma are detected by examining
the amount of a PD-1 polypeptide in a test sample (i.e., a patient
derived sample). A change in the level if the PD-1 polypeptide
compared to a control sample is indicative of cancer in the
subject. The change may be an increase or a decrease in the PD-1
polypeptide relative to a control sample. The control sample is
prepared (i.e., fractionated) in a similar fashion as the test
sample.
[0137] A sample is for example, blood, serum, acsites fluid, urine,
or other bodily fluids. Preferably the sample is a T-cell or a
B-cell
[0138] The amount of the PD-1 is determined in the test sample and
compared to the expression of the normal control level. By normal
control level is meant the expression level of a PD-1 polypeptide
typically found in a subject not suffering from a cancer. An
increase of the level in the patient derived sample of a PD-1
indicates that the subject is suffering from or is at risk of
developing cancer. In contrast, when the methods are applied
prophylacticly, a similar level or a decrease in the level in the
patient derived sample of a PD-1 polypeptide indicates that the
subject is not suffering from or is at risk of developing cancer.
An increase of the level in the patient derived sample of a PD-1
polypeptide indicates that the subject is suffering from or is at
risk of developing cancer.
[0139] The alteration in the amount of the PD-1 polypeptide is
statistically significant. By statistically significant is meant
that the alteration is greater than what might be expected to
happen by change alone. Statistical significance is determined by
method known in the art. For example statistical significance is
determined by p-value. The p-values is a measure of probability
that a difference between groups during an experiment happened by
chance. (P(z.gtoreq.z.sub.observed)). For example, a p-value of
0.01 means that there is a 1 in 100 chance the result occurred by
chance. The lower the p-value, the more likely it is that the
difference between groups was caused by treatment. An alteration is
statistically significant if the p-value is at least 0.05.
Preferably, the p-value is 0.04, 0.03, 0.02, 0.01, 0.005, 0.001 or
less.
[0140] The "diagnostic accuracy" of a test, assay, or method
concerns the ability of the test, assay, or method to distinguish
between patients having cancer or at risk for cancer is based on
whether the patients have a "clinically significant presence" of a
PD-1 polypeptide. By "clinically significant presence" is meant
that the presence of the PD-1 polypeptide in the patient (typically
in a sample from the patient) is higher or lower than the
predetermined cut-off point (or threshold value) for that PD-1
polypeptide and therefore indicates that the patient has cancer for
which the sufficiently high presence of that protein is a
marker.
[0141] The terms "high degree of diagnostic accuracy" and "very
high degree of diagnostic accuracy" refer to the test or assay for
that PD-1 polypeptide with the predetermined cut-off point
correctly (accurately) indicating the presence or absence of the
cancer. A perfect test would have perfect accuracy. Thus, for
individuals who have diabetes, the test would indicate only
positive test results and would not report any of those individuals
as being "negative" (there would be no "false negatives"). In other
words, the "sensitivity" of the test (the true positive rate) would
be 100%. On the other hand, for individuals who did not have
diabetes, the test would indicate only negative test results and
would not report any of those individuals as being "positive"
(there would be no "false positives"). In other words, the
"specificity" (the true negative rate) would be 100%. See, e.g.,
O'Marcaigh A S, Jacobson R M, "Estimating The Predictive Value Of A
Diagnostic Test, How To Prevent Misleading Or Confusing Results,"
Clin. Ped. 1993, 32(8): 485-491, which discusses specificity,
sensitivity, and positive and negative predictive values of a test,
e.g., a clinical diagnostic test.
[0142] Changing the cut point or threshold value of a test (or
assay) usually changes the sensitivity and specificity but in a
qualitatively inverse relationship. For example, if the cut point
is lowered, more individuals in the population tested will
typically have test results over the cut point or threshold value.
Because individuals who have test results above the cut point are
reported as having the disease, condition, or syndrome for which
the test is being run, lowering the cut point will cause more
individuals to be reported as having positive results (i.e., that
they have cancer). Thus, a higher proportion of those who have
cancer will be indicated by the test to have it. Accordingly, the
sensitivity (true positive rate) of the test will be increased.
However, at the same time, there will be more false positives
because more people who do not have the disease, condition, or
syndrome (i.e., people who are truly "negative") will be indicated
by the test to have PD-1 polypeptide values above the cut point and
therefore to be reported as positive (i.e., to have the disease,
condition, or syndrome) rather than being correctly indicated by
the test to be negative. Accordingly, the specificity (true
negative rate) of the test will be decreased. Similarly, raising
the cut point will tend to decrease the sensitivity and increase
the specificity. Therefore, in assessing the accuracy and
usefulness of a proposed medical test, assay, or method for
assessing a patient's condition, one should always take both
sensitivity and specificity into account and be mindful of what the
cut point is at which the sensitivity and specificity are being
reported because sensitivity and specificity may vary significantly
over the range of cut points.
[0143] There is, however, an indicator that allows representation
of the sensitivity and specificity of a test, assay, or method over
the entire range of cut points with just a single value. That
indicator is derived from a Receiver Operating Characteristics
("ROC") curve for the test, assay, or method in question. See,
e.g., Shultz, "Clinical Interpretation Of Laboratory Procedures,"
chapter 14 in Teitz, Fundamentals of Clinical Chemistry, Burtis and
Ashwood (eds.), 4th edition 1996, W.B. Saunders Company, pages
192-199; and Zweig et al., "ROC Curve Analysis: An Example Showing
The Relationships Among Serum Lipid And Apolipoprotein
Concentrations In Identifying Patients With Coronory Artery
Disease," Clin. Chem., 1992, 38(8): 1425-1428.
[0144] An ROC curve is an x-y plot of sensitivity on the y-axis, on
a scale of zero to one (i.e., 100%), against a value equal to one
minus specificity on the x-axis, on a scale of zero to one (i.e.,
100%). In other words, it is a plot of the true positive rate
against the false positive rate for that test, assay, or method. To
construct the ROC curve for the test, assay, or method in question,
patients are assessed using a perfectly accurate or "gold standard"
method that is independent of the test, assay, or method in
question to determine whether the patients are truly positive or
negative for the disease, condition, or syndrome (for example,
coronary angiography is a gold standard test for the presence of
coronary atherosclerosis). The patients are also tested using the
test, assay, or method in question, and for varying cut points, the
patients are reported as being positive or negative according to
the test, assay, or method. The sensitivity (true positive rate)
and the value equal to one minus the specificity (which value
equals the false positive rate) are determined for each cut point,
and each pair of x-y values is plotted as a single point on the x-y
diagram. The "curve" connecting those points is the ROC curve.
[0145] The area under the curve ("AUC") is the indicator that
allows representation of the sensitivity and specificity of a test,
assay, or method over the entire range of cut points with just a
single value. The maximum AUC is one (a perfect test) and the
minimum area is one half. The closer the AUC is to one, the better
is the accuracy of the test.
[0146] By a "high degree of diagnostic accuracy" is meant a test or
assay (such as the test of the invention for determining the
clinically significant presence of PD-1 polypeptide, which thereby
indicates the presence of diabetes) in which the AUC (area under
the ROC curve for the test or assay) is at least 0.70, desirably at
least 0.75, more desirably at least 0.80, preferably at least 0.85,
more preferably at least 0.90, and most preferably at least
0.95.
[0147] By a "very high degree of diagnostic accuracy" is meant a
test or assay in which the AUC (area under the ROC curve for the
test or assay) is at least 0.875, desirably at least 0.90, more
desirably at least 0.925, preferably at least 0.95, more preferably
at least 0.975, and most preferably at least 0.98.
[0148] Optionally, expression of other known biomarkers for a
particular cancer are also determined as further indication of
whether or not the subject is carrying a cancer. For example, CD10,
bcl-6, CD20, CD57 or CXCR5 is detected.
[0149] The PD-1 polypeptide and the additional biomarkers are
detected in any suitable manner, but are typically detected by
contacting a sample from the patient with an antibody which binds
the PD-1 or biomarker and then detecting the presence or absence of
a reaction product. The antibody may be monoclonal, polyclonal,
chimeric, or a fragment of the foregoing, as discussed in detail
above, and the step of detecting the reaction product may be
carried out with any suitable immunoassay. The sample from the
subject is typically a biological fluid as described above, and may
be the same sample of biological fluid used to conduct the method
described above.
[0150] Expression of a PD-1 polypeptide also allows for the course
of treatment of cancer to be monitored. In this method, a
biological sample is provided from a subject undergoing treatment,
e.g., surgical, chemotherapeutic or hormonal treatment, for a
cancer. If desired, biological samples are obtained from the
subject at various time points before, during, or after treatment.
Expression of a PD-1 is then determined and compared to a
reference, e.g. control whose cancer state is known. The reference
sample has been exposed to the treatment. Alternatively, the
reference sample has not been exposed to the treatment. Optionally,
such monitoring is carried out preliminary to a second look
surgical surveillance procedures and subsequent surgical
surveillance procedures. For example, samples may be collected from
subjects who have received initial surgical treatment for cancer
and subsequent treatment with anti-neoplastic agents for that
cancer to monitor the progress of the treatment.
[0151] If the reference sample is from a subject that does not have
cancer, a similarity or a decrease in the amount of the PD-1
polypeptide in the test sample and the reference sample indicates
that the treatment is efficacious. However, an increase in the
amount of the PD-1 polypeptide in the test sample and the reference
sample indicates a less favorable clinical outcome or
prognosis.
[0152] By "efficacious" is meant that the treatment leads to a
decrease in the amount of a PD-1 polypeptide, or a decrease in
size, prevalence, or metastatic potential of a tumor in a subject.
Assessment of cancer is made using standard clinical protocols.
Efficacy is determined in association with any known method for
diagnosing or treating the particular tumor. Expression of a PD-1
polypeptide also allows the identification of patients who will be
responsive to systemic, e.g., chemotherapeutic, hormonal or
radiation therapy. In this method, a biological sample is provided
from a subject prior to undergoing surgical treatment, for a
cancer. Expression of a PD-1 polypeptide is then determined and
compared to a biological sample obtained from the subject after
surgical removal of the cancer. The patient will likely be
responsive to systemic treatment if the amount of the PD-1
polypeptide decreases after surgical removal the cancer. In
contrast a the patient will likely not be responsive to systemic
treatment if the amount of the polypeptide remains constant or
increase after surgical removal of the cancer.
[0153] Expression of the PD-1 polypeptide or other cancer
biomarkers is determined at the protein or nucleic acid level using
any method known in the art. For example, Northern hybridization
analysis using probes which specifically recognize one or more of
these sequences can be used to determine gene expression.
Alternatively, expression is measured using
reverse-transcription-based PCR assays, e.g., using primers
specific for the differentially expressed sequence of genes.
Expression is also determined at the protein level, i.e., by
measuring the levels of peptides encoded by the gene products
described herein, or activities thereof. Such methods are well
known in the art and include, e.g., immunoassays based on
antibodies to proteins encoded by the genes. Any biological
material can be used for the detection/quantification of the
protein or it's activity. Alternatively, a suitable method can be
selected to determine the activity of proteins encoded by the
marker genes according to the activity of each protein
analyzed.
[0154] The subject is preferably a mammal. The mammal is, e.g., a
human, non-human primate, mouse, rat, dog, cat, horse, or cow.
Subjects are typically human females or human males The subject has
been previously diagnosed as carrying a cancer, and possibly has
already undergone treatment for the cancer. Alternatively, the
subject has not been previously diagnosis as carrying a cancer. The
present invention is useful with all patients at risk for a cancer.
Although each type of cancer has its own set of risk factors, the
risk of developing cancer increases as with aged, gender, race and
personal and family medical history. Other risk factors are largely
related to lifestyle choices, while certain infections,
occupational exposures and some environmental factors can also be
related to developing cancer.
[0155] Diagnosis of cancer is typically made through the
identification of a mass on an examination, though it may also be
through other means such as a radiological diagnosis, or
ultrasound. Treatment is typically through cytoreductive surgery,
followed by treatment with antineoplastic agents such as docetaxel,
vinorelbine gemcitabine, capecitabine or a combinations of
cyclophosphamide, methotrexate, and fluorouracil; cyclophosphamide,
doxorubicin, and fluorouracil; doxorubicin and cyclophosphamide;
doxorubicin and cyclophosphamide with paclitaxel; doxorubicin
followed by CMF; or Cyclophosphamide, epirubicin and fluorouracil.
In addition, many patients will require radiation therapy.
[0156] Immunoassays carried out in accordance with the present
invention may be homogeneous assays or heterogeneous assays. In a
homogeneous assay the immunological reaction usually involves the
specific antibody (e.g., PD-1 polypeptide), a labeled analyte, and
the sample of interest. The signal arising from the label is
modified, directly or indirectly, upon the binding of the antibody
to the labeled analyte. Both the immunological reaction and
detection of the extent thereof are carried out in a homogeneous
solution. Immunochemical labels which may be employed include free
radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages,
or coenzymes.
[0157] In a heterogeneous assay approach, the reagents are usually
the sample, the antibody, and means for producing a detectable
signal. Samples as described above may be used. The antibody is
generally immobilized on a support, such as a bead, plate or slide,
and contacted with the specimen suspected of containing the antigen
in a liquid phase. The support is then separated from the liquid
phase and either the support phase or the liquid phase is examined
for a detectable signal employing means for producing such signal.
The signal is related to the presence of the analyte in the sample.
Means for producing a detectable signal include the use of
radioactive labels, fluorescent labels, or enzyme labels. For
example, if the antigen to be detected contains a second binding
site, an antibody which binds to that site can be conjugated to a
detectable group and added to the liquid phase reaction solution
before the separation step. The presence of the detectable group on
the solid support indicates the presence of the antigen in the test
sample. Examples of suitable immunoassays are radioimmunoassays,
immunofluorescence methods, or enzyme-linked immunoassays.
[0158] Those skilled in the art will be familiar with numerous
specific immunoassay formats and variations thereof, which may be
useful for carrying out the method disclosed herein. See generally
E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton,
Fla.); see also U.S. Pat. No. 4,727,022 to Skold et al. titled
"Methods for Modulating Ligand-Receptor Interactions and their
Application," U.S. Pat. No. 4,659,678 to Forrest et al. titled
"Immunoassay of Antigens," U.S. Pat. No. 4,376,110 to David et al.,
titled "Immunometric Assays Using Monoclonal Antibodies," U.S. Pat.
No. 4,275,149 to Litman et al., titled "Macromolecular Environment
Control in Specific Receptor Assays," U.S. Pat. No. 4,233,402 to
Maggio et al., titled "Reagents and Method Employing Channeling,"
and U.S. Pat. No. 4,230,767 to Boguslaski et al., titled
"Heterogeneous Specific Binding Assay Employing a Coenzyme as
Label." Antibodies are conjugated to a solid support suitable for a
diagnostic assay (e.g., beads, plates, slides or wells formed from
materials such as latex or polystyrene) in accordance with known
techniques, such as precipitation. Antibodies as described herein
may likewise be conjugated to detectable groups such as radiolabels
(e.g., 35 S, 125 I, 131 I), enzyme labels (e.g., horseradish
peroxidase, alkaline phosphatase), and fluorescent labels (e.g.,
fluorescein) in accordance with known techniques.
[0159] Diagnostic kits for carrying out the methods described
herein are produced in a number of ways. In one embodiment, the
diagnostic kit comprises (a) an antibody (e.g., PD-1 polypeptide)
conjugated to a solid support and (b) a second antibody of the
invention conjugated to a detectable group. The reagents may also
include ancillary agents such as buffering agents and protein
stabilizing agents, e.g., polysaccharides and the like. The
diagnostic kit may further include, where necessary, other members
of the signal-producing system of which system the detectable group
is a member (e.g., enzyme substrates), agents for reducing
background interference in a test, control reagents, apparatus for
conducting a test, and the like. Alternatively, a test kit contains
(a) an antibody, and (b) a specific binding partner for the
antibody conjugated to a detectable group. Ancillary agents as
described above may likewise be included. The test kit may be
packaged in any suitable manner, typically with all elements in a
single container along with a sheet of printed instructions for
carrying out the test.
[0160] This invention is based in part on the experiments described
in the following examples. These examples are provided to
illustrate the invention and should not be construed as
limiting.
Example 1: Inhibition of the PD-1 Pathway in Chronically-Infected
Mice Using Anti-PD-L1 Antibodies
[0161] Mice infected with various strains of the lymphocytic
choriomeningitis virus (LCMV) were used to study the effect of
chronic viral infection on CD8.sup.+ T cell function. The LCMV
Armstrong strain causes an acute infection that is cleared within 8
days, leaving behind a long-lived population of highly functional,
resting memory CD8.sup.+ T cells. The LCMV C1-13 strain, in
contrast, establishes a persistent infection in the host,
characterized by a viremia that lasts up to 3 months. The virus
remains in some tissues indefinitely and antigen specific CD8.sup.+
T cells become functionally impaired. D.sup.bNP396-404 CD8.sup.+ T
cells are physically deleted, while D.sup.bGP33-41 and
D.sup.bGP276-286 CD8.sup.+ T cells persist but lose the ability to
proliferate or secrete anti-viral cytokines, such as IFN-.gamma.
and TNF-.alpha..
[0162] C57BL/6 mice were purchased from the National Cancer
Institute (Frederick, Md.). Mice were infected i.v. with
2.times.10.sup.6 pfu of LCMV-Cl-13. CD4 depletions were performed
by injecting 500 .mu.g of GK1.5 in PBS the day of infection and the
day following the infection. LCMV immune mice are generated by
infecting mice i.p. with 2.times.10.sup.5 pfu LCMV Armstrong.
[0163] Gene array analysis was performed on FACS-purified naive
D.sup.bGP33-41 specific P14 transgenic CD8.sup.+ T cells,
D.sup.bGP33-41 specific memory CD8.sup.+ T cells derived from LCMV
Armstrong immune mice, and D.sup.bGP33-41 specific or
D.sup.bGP276-286 specific CD8.sup.+ T cells derived from CD4.sup.+
depleted LCMV C1-13 infected mice. RNA isolation and gene array
analysis were performed as described in Kaech et al., (Cell
111:837-51, 2002). PD-1 mRNA was highly expressed in exhausted
CD8.sup.+ T cells relative to memory CD8.sup.+ T cells (FIG. 1A).
Furthermore, PD-1 was expressed on the surface of CD8.sup.+ T cells
in LCMV C1-13 infected mice, but was not present on the surface of
CD8.sup.+ T cells after clearance of LCMV Armstrong (FIG. 1B).
Chronically infected mice also expressed higher levels of one of
the ligands of PD-1, PD-L1, on most lymphocytes and APC compared to
uninfected mice. Thus, viral antigen persistence and CD8.sup.+ T
cell exhaustion are concomitant with an induction in PD-1
expression.
[0164] To test the hypothesis that blocking the PD-1/PD-L1 pathway
may restore T cell function and enhance viral control during
chronic LCMV infection, the PD-1/PD-L1 co-inhibitory pathway was
disrupted during chronic LCMV infection using .alpha.PD-L1 blocking
antibodies. A blocking monoclonal antibody against PD-L1 was
administered i.p. every third day to mice infected with LCMV C1-13
(200 .mu.g of rat anti-mouse PD-L1 IgG2b monoclonal antibodies
(clone 10F.5C5 or 10F.9G2)) from day 23 to day 37 post-infection.
At day 37, there was approximately 2.5 fold more D.sup.bNP396-404
specific CD8.sup.+ T cells and 3 fold more D.sup.bGP33-41 specific
CD8.sup.+ T cells in treated mice relative to the untreated
controls (FIG. 2A). The induction in proliferation was specific to
CD8.sup.+ T cells since the number of CD4+ T cells in the spleen
were approximately the same in both treated mice and untreated mice
(.about.6.times.10.sup.4 IA.sup.bGP61-80 of CD4+ T cells per
spleen).
[0165] In addition to an increase in CD8+ T cell proliferation, the
inhibition of PD-1 signaling also resulted in an increased
production of anti-viral cytokines in virus-specific CD8.sup.+ T
cells. The production of IFN-.gamma. and TNF-.alpha. by CD8+ T
cells to eight different CTL epitopes was determined. The combined
response was 2.3 fold higher in treated mice as compared to
untreated mice (FIGS. 2B and 2C). A 2-fold increase in the
frequency of TNF-.alpha. producing cells was also observed
following treatment (FIG. 2D). Viral clearance was also accelerated
as the virus was cleared from the serum, spleen, and liver of
treated mice. Reduced viral titers were observed in the lung and
kidney (.about.10 fold) by day 37 post-infection (14 days following
initiation of treatment) in treated mice. Untreated mice, however,
displayed significant levels of virus in all these tissues (FIG.
2E). Viral titers in serum and tissue homogenates were determined
using Vero cells, as described in Ahmed et al. (J. Virol. 51:34-41,
1984). The results showing that a PD-1 inhibitor increases CD8+ T
cell proliferation and viral clearance therefore indicate that the
inhibition of PD-1 signaling restores CD8+ T cell function.
Furthermore, inhibition of PD-1 signaling also enhanced B cell
responses as the number of LCMV specific antibody secreting cells
in the spleen was also increased (>10-fold) following
treatment.
[0166] CD4.sup.+ T cells play a key role in the generation and
maintenance of CD8+ T cell responses. In this regard, CD8+ T cells
primed in the absence of CD4+ T cell (so-called "helpless" CD8+ T
cells) are incapable of mounting normal immune responses.
Furthermore, chronic LCMV infection is more severe in the absence
of CD4+ T cells. Accordingly, helpless T cells generated during
LCMV-Cl-13 infection display an even more profound functional
impairment than T cells generated in the presence of CD4+ T cells.
D.sup.bNP396-404 specific CD8.sup.+ T cells are deleted to
undetectable levels, and D.sup.bGP33-41 and D.sup.bGP276-286
CD8.sup.+ T cells completely lose the ability to secrete
IFN-.gamma. and TNF-.alpha..
[0167] CD4.sup.+ T cells were depleted at the time of LCMV-Cl-13
infection and mice were treated with anti-PD-L antibodies treatment
from day 46 to day 60 post-infection. LCMV-specific CD4.sup.+ T
cells were not detectable by intracellular IFN-.gamma. staining
before or after treatment. Following treatment, treated mice had
approximately 7 fold more D.sup.bGP276-286 CD8.sup.+ T cells and 4
fold more D.sup.bGP33-41 CD8.sup.+ T cells in their spleen than
untreated control mice (FIG. 3A). The number of virus-specific
CD8.sup.+ T cells in the spleen was also increased (FIG. 3B). This
increase in virus-specific CD8.sup.+ T cells in treated mice was
attributed to an increase in proliferation, as detected by BrdU
incorporation. 43% of D.sup.bGP276-286 CD8.sup.+ T cells
incorporated intermediate levels of BrdU and 2% incorporated high
levels of BrdU in untreated mice, while 50% D.sup.bGP276-286
CD8.sup.+ T cells incorporated intermediate levels of BrdU and 37%
incorporated high levels of BrdU in treated mice. BrdU analysis was
performed by introducing 1 mg/ml BrdU in the drinking water during
treatment and staining was performed according to the
manufacturer's protocol (BD Biosciences, San Diego, Calif.).
Moreover, treated mice contained a higher percentage of CD8.sup.+ T
cells expressing the cell cycle-associated protein Ki67 (60% versus
19% in untreated mice, FIG. 3C). Response to treatment in CD8.sup.+
T cells in the PBMC was restricted to mice having high levels of
CD8.sup.+ T cell expansion.
[0168] PD-1 inhibition also increased anti-viral cytokine
production in helpless, exhausted virus-specific CD8.sup.+ T cells.
Following treatment, the number of D.sup.bGP33-41 and
D.sup.bGP276-286 CD8.sup.+ T cells that produce IFN-.gamma. was
markedly increased (FIG. 4A), though higher numbers of
D.sup.bNP396-404, K.sup.bNP205-212, D.sup.bNP166-175, and
D.sup.bGP92-101 specific CD8+ T cells were also detected in treated
mice (FIG. 4A). 50% of D.sup.bGP276-286 specific CD8.sup.+ T cells
from treated mice can produce IFN-.gamma. compared to the 20% of
D.sup.bGP276-286 specific CD8.sup.+ T cells in control untreated
mice. (FIG. 4B). Levels of IFN-.gamma. and TNF-.alpha. produced by
D.sup.bGP276-286 specific CD8.sup.+ T cells from treated mice,
however, were lower than fully functional D.sup.bGP276-286 specific
memory cells. (FIG. 4C).
[0169] PD-1 inhibition also increased the lytic activity of
helpless, exhausted virus-specific CD8.sup.+ T cells. Ex vivo lytic
activity of virus-specific CD8.sup.+ T cells was detected following
treatment, using a .sup.51Cr release assay (Wherry et al., 2003. J.
Virol. 77:4911-27). Viral titers were reduced by approximately 3
fold in the spleen, 4 fold in the liver, 2 fold in the lung, and 2
fold in serum after 2 weeks of treatment relative to untreated
mice. (FIG. 4E).
[0170] These results therefore demonstrate that blocking the PD-1
pathway breaks CTL peripheral tolerance to a chronic viral
infection, and that exhausted CD8.sup.+ T cells deprived of
CD4.sup.+ T cell help are not irreversibly inactivated.
Example 2: Administration of Anti-Viral Vaccine and PD-1
Inhibitor
[0171] One approach for boosting T cell responses during a
persistent infection is therapeutic vaccination. The rationale for
this approach is that endogenous antigens may not be presented in
an optimal or immunogenic manner during chronic viral infection and
that providing antigen in the form of a vaccine may provide a more
effective stimulus for virus-specific T and B cells. Using the
chronic LCMV model, mice were administered a recombinant vaccinia
virus expressing the LCMV GP33 epitope as a therapeutic vaccine
(VVGP33), which resulted in a modest enhancement of CD8+ T cell
responses in some chronically infected mice Four out of the nine
chronically infected mice that received the therapeutic vaccine
showed a positive response while none of the control mice had a
significant increase in the immune response against GP33. When this
therapeutic vaccination was combined with a PD-L1 inhibitor, LCMV
specific T cell responses were boosted to a greater level than
compared to either treatment alone and the effect of combined
treatment was more than additive.
Example 3: Inhibition of the PD-1 Pathway in Chronically-Infected
Mice Using PD-1 RNAi
[0172] RNA interference (RNAi) is capable of silencing gene
expression in mammalian cells. Long double stranded RNAS (dsRNAs)
are introduced into cells and are next processed into smaller,
silencing RNAs (siRNAs) that target specific mRNA molecules or a
small group of mRNAs. This technology is particularly useful in
situations where antibodies are not functional. For example, RNAi
may be employed in a situation in which unique splice variants
produce soluble forms of PD-1 and CTLA-4.
[0173] PD-1 silencer RNAs are inserted into a commercially
available siRNA expression vector, such as pSilencer.TM. expression
vectors or adenoviral vectors (Ambion, Austin, Tex.). These vectors
are then contacted with target exhausted T cells in vivo or ex vivo
(See, Example 4).
Example 4: Ex Vivo Rejuvenation of Exhausted T Cells
[0174] Virus-specific exhausted CD8.sup.+ T cells are isolated from
LCMV-Cl-13 chronically infected mice using magnetic beads or
density centrifugation. Transfected CD8.sup.+ T cells are contacted
with a monoclonal antibody that targets PD-L1, PD-L2 or PD-1. As
described in Example 1, inhibition of the PD-1 pathway results in
the rejuvenation of the CD8.sup.+ T cells. Accordingly, there is an
increase in CD8+ T cell proliferation and cytokine production, for
example. These rejuvenated CD8.sup.+ T cells are reintroduced into
the infected mice and viral load is measured as described in
Example 1.
Example 5: In Vitro Screening of Novel CD8.sup.+ T Cell Rejuvenator
Compounds
[0175] Compounds that modulate the PD-1 pathway can be identified
in in vivo and ex vivo screening assays based on their ability to
reverse CD8.sup.+ T cell exhaustion resulting from chronic viral
infection.
[0176] Exhausted CD8.sup.+ T cells are derived from mice
chronically infected with LCMV-Cl-13 and next contacted with a test
compound. The amount of anti-viral cytokines (e.g., IFN-.gamma. or
TNF-.alpha.) released from the contacted T cell is measured, for
example, by ELISA or other quantitative method, and compared to the
amount, if any, of the anti-viral cytokine released from the
exhausted T cell not contacted with the test compound. An increase
in the amount of anti-viral cytokine released by treated cells
relative to such amount in untreated cells identifies the compound
as a PD-1 inhibitor, useful to modulate T cell activity.
Example 6: In Vivo Screening of Novel CD8.sup.+ T Cell Rejuvenator
Compounds
[0177] Exhausted CD8.sup.+ T cells are derived from mice
chronically infected with LCMV-Cl-13.
[0178] A test compound is administered intravenously to the
infected mice. The amount of anti-viral cytokines (e.g.,
IFN-.gamma. or TNF-.alpha.) that is released into the serum of
treated and untreated mice is measured, for example, by ELISA or
other quantitative method, and compared. An increase in the amount
of anti-viral cytokine found in the serum in treated mice relative
to such amount in untreated mice identifies the test compound as a
PD-1 inhibitor. Alternatively, the viral titer (e.g., serum viral
titer) can be determined prior and subsequent to treatment of the
test compound.
Example 7: Chimpanzees as a Model for Immunotherapy of Persistent
HCV Infection
[0179] Chimpanzees provide a model of HCV persistence in humans.
Defects in T cell immunity leading to life-long virus persistence
both include a deficit in HCV-specific CD4+T helper cells and
impaired or altered CD8+T effector cell activity. Persistently
infected chimpanzees are treated with antibodies against CTLA-4,
PD-1, or a combination of the two. The efficacy of blockade of the
inhibitory pathways, combined with vaccination using recombinant
structural and non-structural HCV proteins, and whether such
strategies can enhance the frequency and longevity of
virus-specific memory T cells are determined. The defect in T cell
immunity is exclusively HCV-specific in persistently infected
humans and chimpanzees. The blood and liver of infected chimpanzees
are examined for expression of CTLA-4, PD-1, BTLA and their ligands
and for the presence of Treg cells. Antiviral activity may then be
restored by delivering to chimpanzees humanized monoclonal
antibodies that block signaling through these molecules.
[0180] Persistently infected chimpanzees are treated with humanized
.alpha.CTLA-4 antibodies (MDX-010, Medarex) or .alpha.PD-1
antibodies. The initial dose of MDX-010 is 0.3 mg/kg followed 2
weeks later by 1.0 mg/kg and then 3, 10, 30 mg/kg at three week
intervals. After treatment with antibodies to co-inhibitory
molecules, the humoral and cellular immune responses as well as the
HCV RNA load will be determined. Samples are collected at weeks 1,
2, 3, 5, and 8, and then at monthly intervals. Samples include: 1)
serum for analysis of transaminases, autoantibodies, neutralizing
antibodies to HCV, and cytokine responses, 2) plasma for viral load
and genome evolution, 3) PBMC for in vitro measures of immunity,
costimulatory/inhibitory receptor expression and function, 4) fresh
(unfixed) liver for isolation of intrahepatic lymphocytes and RNA,
and 5) fixed (formalin/paraffin embedded) liver for histology and
immunohistochemical analysis. Regional lymph nodes are also
collected at 2 or 3 time points to assess expression of
co-inhibitory molecules and splice variants by immunohistochemistry
and molecular techniques. Assays to evaluate the efficacy and
safety of these therapies safety will be performed as described
herein.
[0181] To determine if vaccination with HCV antigens potentiates
the therapeutic effect of antibodies to PD-1, chimpanzees are
treated as follows: 1) intramuscular immunization with recombinant
envelope glycoproteins E1 and E2 (in MF59 adjuvant) and other
proteins (core plus NS 3, 4, and 5 formulated with ISCOMS) at weeks
0, 4, and 24; 2) intramuscular immunization with the vaccine used
in 1) but co-administered with .alpha.CTLA-4 antibodies (30 mg of
each/Kg body weight, intravenously at weeks 0, 4, and 24 when
vaccine is given); 3) identical to 2) except that .alpha.PD-1 (or
BTLA) antibodies are substituted for the CTLA-4 antibodies; 4)
identical to Groups 2 and 3 except that a combination of CTLA-4 and
PD-1 (or BTLA) antibodies are used in addition to the vaccine.
HCV-specific T and B cell responses are monitored at monthly
intervals after immunization for a period of 1 year.
[0182] Markers examined on HCV-tetramer+ and total T cells in this
analysis include markers of differentiation (e.g. CD45RA/RO, CD62L,
CCR7, and CD27), activation (e.g. CD25, CD69, CD38, and HLA-DR),
survival/proliferation (e.g. bcl-2 and Ki67), cytotoxic potential
(e.g. granzymes and perforin), and cytokine receptors (CD122 and
CD127). An interesting correlation exists between pre-therapy
levels of the chemokine IP-10 and response to PEG
IFN-.gamma./ribavirin. IP-10 levels are measured to investigate a
potential correlation between negative regulatory pathways or
HCV-specific T cell responses and IP-10 levels. Expression of
inhibitory receptors and ligands on PBMC are performed by flow
cytometry.
Example 8: PD-1 Immunostaining in Reactive Lymphoid Tissue
Materials
[0183] Case material was obtained from the Brigham & Women's
Hospital, Boston, Mass., in accordance with institutional policies.
All diagnoses were based on the histologic and immunophenotypic
features described in the World Health Organization Lymphoma
Classification system (Jaffe E S, et al. 2001) and in all cases
diagnostic material was reviewed by a hematopathologist.
Immunostaining
[0184] Immunostaining for PD-1 was performed on formalin-fixed
paraffin embedded tissue sections following microwave antigen
retrieval in 10 mM citrate buffer, pH 6.0 with a previously
described anti-human PD-1 monoclonal antibody (2H7; 5), using a
standard indirect avidin-biotin horseradish peroxidase method and
diaminobenzidine color development, as previously described (Jones
D, et al. 1999; Dorfman D M, et al. 2003). Cases were regarded as
immunoreactive for PD-1 if at least 25% of neoplastic cells
exhibited positive staining. PD-1 staining was compared with that
of mouse IgG isotype control antibody diluted to identical protein
concentration for all cases studied, to confirm staining
specificity.
[0185] Monoclonal antibody 2H7 for PD-1 was used to stain
formalin-fixed, paraffin-embedded specimens of reactive lymphoid
tissue, thymus, and a range of cases of B cell and T cell
lymphoproliferative disorders. In specimens of tonsil exhibiting
reactive changes, including follicular hyperplasia, a subset of
predominantly small lymphocytes in the germinal centers exhibited
cytoplasmic staining for PD-1, with infrequent PD-1-positive cells
seen in the interfollicular T cell zones. The PD-1 staining pattern
in germinal centers was virtually identical to that seen with an
antibody to CD3, a pan-T cell marker, whereas an antibody to CD20,
a pan-B cell marker, stained the vast majority of germinal center B
cells. Similar results were seen in histologic sections of reactive
lymph node and spleen. No PD-1 staining was observed in adult
thymus.
Example 9: PD-1 Immunostaining in Paraffin Embedded Tissue Sections
of B Cell and T Cell Lymphoproliferative Disorders
[0186] A range of B cell and T cell lymphoproliferative disorders
for PD-1 expression were studied; the results are summarized in
Table 1. Forty-two cases of B cell lymphoproliferative disorders
were examined for PD-1 expression, including representative cases
of precursor B lymphoblastic leukemia/lymphoblastic lymphoma, as
well as a range of lymphoproliferative disorders of mature B cells,
including a number of B cell non-Hodgkin lymphomas of follicular
origin, including 6 cases of follicular lymphoma and 7 cases of
Burkitt lymphoma. None of the B cell lymphoproliferative disorders
showed staining for PD-1. In some cases, non-neoplastic reactive
lymphoid tissue was present, and showed a PD-1 staining pattern as
seen in tonsil and other reactive lymphoid tissue noted above.
[0187] Similarly, in 25 cases of Hodgkin lymphoma, including 11
cases of classical Hodgkin lymphoma and 14 case of lymphocyte
predominant Hodgkin lymphoma, the neoplastic cells did not exhibit
staining for PD-1. Interestingly, in all 14 cases of lymphocyte
predominant Hodgkin lymphoma, the T cells surrounding neoplastic
CD20-positive L&H cells were immunoreactive for PD-1, similar
to the staining pattern noted for CD57+ T cells in lymphocyte
predominant Hodgkin lymphoma. These PD-1-positive cells were a
subset of the total CD3+ T cell population present.
[0188] A range of T cell lymphoproliferative disorders were studied
for expression of PD-1; the results are summarized in Table 1.
Cases of precursor T cell lymphoblastic leukemia/lymphoblastic
lymphoma, a neoplasm of immature T cells of immature T cells, were
negative for PD-1, as were neoplasms of peripheral, post-thymic T
cells, including cases of T cell prolymphocytic leukemia,
peripheral T cell lymphoma, unspecified, anaplastic large cell
lymphoma, and adult T cell leukemia/lymphoma. In contrast, all 19
cases of angioimmunoblastic lymphoma contained foci of
PD-1-positive cells that were also immunoreactive for pan-T cell
markers such as CD3. PD-1-positive cells were consistently found at
foci of expanded CD21+ follicular dendritic cells (FDC) networks, a
characteristic feature of angioimmunoblastic lymphoma.
TABLE-US-00001 TABLE 1 PD-1 immunostaining in lymphoproliferative
disorders. PD-1 immunostaining B cell LPDs 0/42* B-LL/LL 0/3 CLL
0/4 MCL 0/4 FL 0/6 MZL 0/3 HCL 0/3 DLBCL 0/6 BL 0/7 LPL 0/3 MM 0/3
Hodgkin lymphoma 0/25 Classical 0/11 Nodular lymphocyte 0/14**
predominant T cell LPDs 18/55 T-LL/LL 0/5 T-PLL 0/3 AIL 19/19 PTCL,
unspecified 0/14 ALCL 0/12 ATLL 0/3 Abbreviations: B-LL/LL -
precursor B cell lymphoblastic lymphoma/lymphoblastic leukemia; CLL
- chronic lymphocytic leukemia; MCL - mantle cell lymphoma; FL -
follicular lymphoma; MZL - marginal zone lymphoma; HCL - hairy cell
leukemia; DLBCL - diffuse large B cell lymphoma; BL - Burkitt
lymphoma; LPL - lymphoplasmacytic lymphoma; MM - multiple myeloma;
T-LL/L - precursor T lymphoblastic leukemia/lymphoblastic lymphoma;
T-PLL- T cell prolymphocytic leukemia; AIL - angioimmunoblastic
lymphoma; PTCL - peripheral T cell lymphoma, unspecified; ALCL -
anaplastic large cell lymphoma; ATLL- adult T cell
leukemia/lymphoma. *number of immunoreactive cases/total number of
cases **PD-1-positive cells form rosettes around neoplastic L&H
cells in 14/14 cases
Example 10: General Methods for Studying PD-1 Expression on
HIV-Specific Human CD8 T Cells
[0189] The following methods were used to perform the experiments
detailed inn Examples 11-14.
Study Subjects
[0190] Study participants with chronic clade C HIV-1 infection were
recruited from outpatient clinics at McCord Hospital, Durban, South
Africa, and St. Mary's Hospital, Mariannhill, South Africa.
Peripheral blood was obtained from 65 subjects in this cohort, all
of whom were antiretroviral therapy naive at the time of analysis.
Subjects were selected for inclusion based on their expressed HLA
alleles matching the ten class I tetramers that were constructed
(see below). The median viral load of the cohort was 42,800 HIV-1
RNA copies/ml plasma (range 163-750,000), and the median absolute
CD4 count was 362 (range 129-1179). Information regarding duration
of infection was not available. All subjects gave written informed
consent for the study, which was approved by local institutional
review boards.
Construction of PD-1 and PD-L1 Antibodies
[0191] Monoclonal antibodies to human PD-L1 (29E.2A3, mouse IgG2b)
and PD-1 (EH12, mouse IgG1) were prepared as previously described
and have been shown to block the PD-1:PD-L1 interaction.
MHC Class I Tetramers
[0192] Ten HIV MHC Class I tetramers, synthesized as previously
described (Altman J D, et al. 1996), were used for this study:
A*0205 GL9 (p24, GAFDLSFFL; SEQ ID NO:), A*3002 KIY9 (Integrase,
KIQNFRVYY; SEQ ID NO:2), B*0801 DI8 (p24, DIYKRWII; SEQ ID NO:3),
B*0801 FL8 (Nef, FLKEKGGL; SEQ ID NO:4), B*4201 RM9 (Nef,
RPQVPLRPM; SEQ ID NO:5), B*4201 TL9 (p24, TPQDLNTML; SEQ ID NO:6),
B*4201 TL10 (Nef, TPGPGVRYPL; SEQ ID NO:7), B*4201 YL9 (RT,
YPGIKVKQL; SEQ ID NO:8), B*8101 TL9 (p24, TPQDLNTML; SEQ ID NO:9),
and Cw0304 YL9 (p24, YVDRFFKTL; SEQ ID NO: 10). The plasmid
constructs expressing A*0205, A*3002, and Cw*0304 were kindly
provided by Drs. Eugene Ravkov and John Altman, NIH Tetramer Core
Facility, Atlanta, Ga.
HLA Class I Tetramer Staining and Phenotypic Analysis
[0193] Freshly isolated peripheral blood mononuclear cells (PBMC,
0.5 million) were stained with tetramer for 20 minutes at
37.degree. C. The cells were then washed once with phosphate
buffered saline (PBS), pelleted, and stained directly with
fluorescein isothiosyanate (FITC)-conjugated anti-CD8 (Becton
Dickinson), phycoerythrin-conjugated anti-PD-1 (clone EH12), and
ViaProbe (Becton Dickinson). Cells were incubated for 20 minutes at
room temperature, washed once in PBS, and resuspended in 200 .mu.l
PBS with 1% paraformaldehyde and acquired on a
fluorescence-activated cell sorter (FACSCalibur, Becton Dickinson).
A minimum of 100,000 events were acquired on the FACSCalibur.
CFSE Proliferation Assays
[0194] One million freshly isolated PBMC were washed twice in PBS,
pelleted, and resuspended in 1 ml of 0.5 .mu.M carboxy-fluorescein
diacetate, succinimidyl ester (CFSE, Molecular Probes) for 7
minutes at 37.degree. C. The cells were washed twice in PBS,
resuspended in 1 ml R10 medium (RPMI 1640 supplemented with
glutiathione, penicillin, streptomycin, and 10% fetal calf serum
[FCS]), and plated into one well of a 24-well plate. Initial
studies revealed that a final concentration of 0.2 .mu.g/ml peptide
yielded optimal proliferative responses, therefore this was the
final peptide concentration in the well used for each assay.
Negative control wells consisted of PBMC in medium alone, or PBMC
in medium with purified anti-PD-L1 (10 .mu.g/ml), and positive
control wells were stimulated with 10 .mu.g/ml of
phytohemagluttinin (PHA). Following 6-day incubation in a
37.degree. C. incubator, the cells were washed with 2 ml PBS and
stained with PE-conjugated MHC Class I tetramers, ViaProbe (Becton
Dickinson), and anti-CD8-APC antibodies. Cells were acquired on a
FACSCalibur and analyzed by CellQuest.RTM. software (Becton
Dickinson). Cells were gated on ViaProbe.sup.- CD8.sup.+
lymphocytes. The fold increase in tetramer.sup.+ cells was
calculated by dividing the percentage of CD8.sup.+ tetramer.sup.+
cells in the presence of peptide by the percentage of CD8.sup.+
tetramer.sup.+ cells in the absence of peptide stimulation.
Statistical Analysis
[0195] Spearman correlation, Mann-Whitney test, and paired t-test
analyses were performed using GraphPad Prism Version 4.0a. All
tests were 2-tailed and p values of p<0.05 were considered
significant.
Example 11: PD-1 Expression on HIV-Specific CD8 T Cells
[0196] A panel of 10 MHC Class I tetramers specific for dominant
HIV-1 clade C virus CD8 T cell epitopes was synthesized, based on
prevalent HLA alleles and frequently targeted epitopes in Gag, Nef,
Integrase, and RT (Kiepiela P, et al. 2004), allowing direct
visualization of surface PD-1 expression on these cells. High
resolution HLA typing was performed on the entire cohort, and a
subset of 65 antiretroviral therapy naive persons was selected for
study based on expression of relevant HLA alleles. A total of 120
individual epitopes were examined, and representative ex vivo
staining of PD-1 on HIV tetramer.sup.+ cells is shown in FIG. 5A.
PD-1 expression was readily apparent on these tetramer.sup.+ cells,
and was significantly higher than in the total CD8 T cell
population from the same individuals (p<0.0001); in turn, PD-1
expression on both tetramer.sup.+ CD8 T cells and the total CD8 T
cell population was significantly higher than in HIV-seronegative
controls (FIG. 5B). For eight of the ten tetramers tested at least
one person was identified in whom the level of expression on
antigen-specific CD8 cells was 100% (FIG. 5C). PBMC from 3 to 25
individuals were stained for each HIV tetramer response, with
median PD-1 expression levels ranging from 68% to 94% of
tetramer.sup.+ cells (FIG. 5C). These findings were further
confirmed by analysis of the mean fluorescence intensity (MFI) of
PD-1 on both tetramer.sup.+ cells and the total CD8 T cell
population (FIG. 5B,C).
[0197] It was next determined whether there was evidence for
epitope-specific differences in terms of PD-1 expression levels in
persons with multiple detectable responses. Of the 65 persons
examined, 16 individuals had between 3 and 5 tetramer positive
responses each. PD-1 expression was nearly identical and
approaching 100% for each response analyzed for three of the
sixteen subjects; however, the other 13 individuals displayed
different patterns of PD-1 expression depending on the epitope
(FIG. 5D). These data indicate that PD-1 expression may be
differentially expressed on contemporaneous epitope-specific CD8 T
cells from a single person, perhaps consistent with recent data
indicating epitope-specific differences in antiviral efficacy
(Tsomides T J, et al. 1994; Yang O, et al. 1996; Loffredo J T, et
al. 2005).
Example 12: The Relationship Between PD-1 Expression and HIV
Disease Progression
[0198] The relationship was determined between PD-1 expression on
HIV-specific CD8 T cells and plasma viral load and CD4 counts, both
of which are predictors of HIV disease progression. Consistent with
previous studies, the relationship between the number of tetramer
positive cells and viral load or CD4 count failed to show any
significant correlation (FIG. 6A, B). In contrast, there were
significant positive correlations with viral load and both the
percentage and MFI of PD-1 expression on HIV tetramer positive
cells (p=0.0013 and p<0.0001, respectively; FIG. 6A). There were
also inverse correlations between CD4 count and both the percentage
and MFI of PD-1 on HIV tetramer positive cells (p=0.0046 and
p=0.0150, respectively; FIG. 6B). Since the tetramers tested likely
represent only a fraction of the HIV-specific CD8 T cell population
in these subjects, the relationship between PD-1 expression on all
CD8 cells and these parameters was also examined. There were
significant positive correlations between viral load and both the
percentage and MFI of PD-1 expression on the total CD8 T cell
population (p=0.0021 and p<0.0001, respectively; FIG. 6C), and
inverse correlations were also observed between CD4 count and both
the percentage and MFI of PD-1 expression on the total CD8 T cell
population (p=0.0049 and p=0.0006, respectively; FIG. 6D). In this
same group, PD-1 expression on CMV-specific CD8 T cells was tested
in 5 subjects, and significantly less PD-1 was expressed on these
cells compared to HIV-specific CD8 T cells (median 23% CMV
tetramer.sup.+ PD-1.sup.+, p=0.0036, data not shown), and was not
different than bulk CD8 T cells in these same individuals,
indicating that high PD-1 expression is not a uniform feature of
all virus-specific CD8 T cells. These data suggest increasing
amounts of antigen in chronic HIV infection result in increased
expression of PD-1 on CD8 T cells, and are consistent with murine
data in chronic LCMV infection, in which PD-1 expression is
associated with functional exhaustion of CD8 T cells (Barber D L,
et al. 2005). Moreover, they provide the first clear association,
in a large study including analysis of multiple epitopes, between
HIV-specific CD8 T cells and either viral load or CD4 count.
Example 13: The Relationship Between PD-1 Expression and CD8 T Cell
Memory Status and Function
[0199] PD-1 expression was next analyzed in the context of a number
of additional phenotypic markers associated with CD8 T cell memory
status and function, including CD27, CD28, CD45RA, CD57, CD62L,
CD127, CCR7, perforin, granzyme B, and Ki67 (FIG. 7).
Representative stainings for these markers on B*4201 TL9
tetramer.sup.+ cells from one individual are shown in FIG. 7A, and
aggregate data for 13 subjects are shown in FIG. 7B. These studies
were limited to those tetramer responses that were greater than 95%
PD-1 positive, as multiparameter flow cytometry of greater than 4
colors was not available in KwaZulu Natal. The HIV tetramer.sup.+
PD-1.sup.+ cells express high levels of CD27 and granzyme B, very
low levels of CD28, CCR7, and intracellular Ki67, low levels of
CD45RA and perforin, and intermediate levels of CD57 and CD62L
(FIG. 7B). These data indicate that HIV-specific PD-1.sup.+ T cells
display an effector/effector memory phenotype, and are consistent
with previous reports of skewed maturation of HIV-specific CD8 T
cells (Champagne P, et al. 2001; Appay V, et al. 2002; Hess C, et
al. 2004). In addition, virus sequencing was performed to determine
whether these cells were driving immune escape (Brown J A, et al.
2003). Of 45 of these tetramer-positive responses evaluated, the
viral epitopes in only 5 were different from the South African
clade C consensus sequence (data not shown), indicating these cells
exert little selection pressure in vivo.
[0200] Previous experiments in mice using the LCMV model showed
that in vivo blockade of PD-1/PD-L1 interaction by infusion of
anti-PD-L1 blocking antibody results in enhanced functionality of
LCMV-specific CD8 T cells as measured by cytokine production,
killing capacity, proliferative capacity, and, most strikingly,
reduction in viral load (Barber D L, et al. 2005).
Example 14: Effect of Blockading the PD-1/PD-L1 Pathway on
Proliferation of HIV-Specific CD8 T Cells
[0201] Because HIV-specific CD8 T cells also exhibit impaired
proliferative capacity (Migueles S A, et al. 2002; Lichterfeld M,
et al. 2004), it was determined whether blockade of the PD-1/PD-L1
could enhance this function in vitro. Representative data from a
B*4201-positive individual are shown in FIG. 8A. Incubation of
freshly isolated CFSE-labeled PBMC with medium alone, or medium
with anti-PD-L1 antibody, resulted in maintenance of a population
of B*4201-TL9-specific CD8 T cells (1.2% of CD8 T cells) that
remained CFSE.sup.hi after six days in culture. Simulation of
CFSE-labeled PBMC for 6 days with TL9 peptide alone resulted in a
4.8-fold expansion of CFSE.sup.lo B*4201 TL9 tetramer+ cells,
whereas stimulation of CFSE-labeled PBMC with TL9 peptide in the
presence of anti-PD-L1 blocking antibody further enhanced
proliferation of TL9-specific cells, resulting in a 10.3-fold
increase in tetramer.sup.+ cells. CFSE proliferation assays were
performed on 28 samples in the presence and absence of purified
anti-human PD-L1 blocking antibody. A significant increase in the
proliferation of HIV-specific CD8.sup.+ T cells was observed in the
presence of peptide plus anti-PD-L1 blocking antibody as compared
to the amount of proliferation following stimulation with peptide
alone (FIG. 8B; p=0.0006, paired t-test). The fold increase of
tetramer.sup.+ cells in the presence of anti-PD-L1 blocking
antibody varied by individual and by epitope within a given
individual (FIG. 8C), again suggesting epitope-specific differences
in the degree of functional exhaustion of these responses.
OTHER EMBODIMENTS
[0202] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
Sequence CWU 1
1
1019PRTArtificial SequenceSynthetic HIV MHC Class I peptide. 1Gly
Ala Phe Asp Leu Ser Phe Phe Leu1 529PRTArtificial SequenceSynthetic
HIV MHC Class I peptide. 2Lys Ile Gln Asn Phe Arg Val Tyr Tyr1
538PRTArtificial SequenceSynthetic HIV MHC Class I peptide. 3Asp
Ile Tyr Lys Arg Trp Ile Ile1 548PRTArtificial SequenceSynthetic HIV
MHC Class I peptide. 4Phe Leu Lys Glu Lys Gly Gly Leu1
559PRTArtificial SequenceSynthetic HIV MHC Class I peptide. 5Arg
Pro Gln Val Pro Leu Arg Pro Met1 569PRTArtificial SequenceSynthetic
HIV MHC Class I peptide. 6Thr Pro Gln Asp Leu Asn Thr Met Leu1
5710PRTArtificial SequenceSynthetic HIV MHC Class I peptide. 7Thr
Pro Gly Pro Gly Val Arg Tyr Pro Leu1 5 1089PRTArtificial
SequenceSynthetic HIV MHC Class I peptide. 8Tyr Pro Gly Ile Lys Val
Lys Gln Leu1 599PRTArtificial SequenceSynthetic HIV MHC Class I
peptide. 9Thr Pro Gln Asp Leu Asn Thr Met Leu1 5109PRTArtificial
SequenceSynthetic HIV MHC Class I peptide. 10Tyr Val Asp Arg Phe
Phe Lys Thr Leu1 5
* * * * *